Note: Descriptions are shown in the official language in which they were submitted.
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ORAL FORMULATIONS OF CANNABIS EXTRACTS AND METHODS OF MAKING
SAME
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to US Provisional
Patent Application
Serial No. 62/884,503 filed on August 8, 2019, the contents of which are
hereby incorporated
by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to oral formulations of cannabinoids,
flavonoids,
terpenes, and other bioactive molecules from plants of the Cannabis genus. The
invention
further relates to the formulation of cannabis extracts with edible oils in
order to enhance the
absorption of orally-administered cannabinoids.
BACKGROUND OF THE INVENTION
[0003] Cannabis has been cultivated and used for medicinal purposes for
thousands of
years (Zuardi, 2006). In our modern era, cannabis use is less commonplace due
to the illegality
of the plant and its components. More recently, cannabis has seen a resurgence
in its use due to
the growing support of medical patients looking to find alternatives to
prescribed
pharmaceutical medications. This has led to several countries adopting a
medical regime for
cannabis cultivation and use, as well as a few countries that have also
adopted a recreational
framework for cannabis use.
[0004] The major bioactive components of the cannabis plant are the
cannabinoids, a class
of molecules that bind to cannabinoid receptors throughout the body. There are
over 120
different cannabinoids present in plants of the Cannabis genus, each with
varying effects on the
body (Aizpurua-Olaizola et al., 2016). The most studied cannabinoids are
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A9tetrahydrocannabinol (THC) and cannabidiol (CBD), the former being known
primarily for
its intoxicating effects, and both being recognized for their medicinal
properties.
[0005] Cannabis can be consumed via a variety of routes and product forms.
Two popular
routes are via inhalation by smoking the flowering parts of the plant
(colloquially known as the
buds of the plant) and by oral ingestion.
[0006] Cannabinoids consumed via the inhalation route of administration
have reported
absorption of 2 to 56% (Huestis, 2007). The large range of bioavailability via
the inhalation
route is due in part to intra- and inter-subject variation in inhalation
dynamics. These include
the number, duration (hold time), and spacing of inhalations, which greatly
influence the degree
of exposure. Comparatively, bioavailability of cannabinoids taken orally is
reported to be quite
low, at 3 to 10% (Huestis, 2007) due the hydrophobic properties of
cannabinoids. The high
lipophilicity of cannabinoids also results in an effect on absorption by the
timing of
consumption relative to fasting or eating, particularly high-fat food (Zgair
et al., 2016), which
could further lead to variation in absorption. The amount of cannabinoids
reaching systemic
circulation is reduced even further as a result of first-pass metabolism
(metabolism of a
drug/molecule by the liver before it reaches the systemic circulation), which
is a common
reason for reduced drug availability following oral consumption (Zgair et al.,
2016). Whether
by inhalation, oral, sublingual or oromucosal administration, absorption of
cannabinoids is
highly variable which can result in issues with predictability of its
physiological effects.
Although cannabinoid absorption following inhalation is superior compared to
other
administration routes, it is still suboptimal, unpredictable and only a small
fraction ultimately
reaches sites of action.
[0007] Due to the health risks associated with smoking and vaporizing, and
the aversion
that most people have to inhalation as a route of administration, orally-
consumed cannabis
products are the preferred product form in the pharmaceutical industry, and
have been
increasing in popularity in the cannabis industry as wellness products. In
addition, oral products
such as tablets, capsules, softgels, etc., are a traditional dosage form
recognized by healthcare
providers, patients, and consumers alike. These products can provide
standardized methods of
dosing, allow for simple directions for use, and can optimize compliance by
patients and use by
consumers due to ease in administration.
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[0008] There therefore exists a need to improve the bioavailability of
cannabinoids from
the gastrointestinal tract so that oral consumption of cannabis products can
approach or exceed
the absorption profile of cannabinoids administered via inhalation without the
negative side
effects associated with smoking or vaporizing.
SUMMARY OF THE INVENTION
[0009] The disclosure provides compositions comprising a cannabis extract
comprising at
least one cannabinoid, and a lipid-based carrier.
[00010] In some embodiments of the compositions of the disclosure, the
lipid-based carrier
comprises omega-3 fatty acids, and at least one of monoacylglycerides,
diacylglycerides,
triglycerides or phospholipids. In some embodiments, the omega-3 fatty acids
comprise omega-
3 monoacylglycerides, omega-3 diacylglycerides, omega-3 phospholipids or a
combination
thereof
[00011] In some embodiments of the compositions of the disclosure, the
lipid based carrier
comprises phospholipids and triglycerides.
[00012] In some embodiments of the compositions of the disclosure, the
lipid based carrier
comprises monoacylglycerides and diacylglycerides.
[00013] In some embodiments of the compositions of the disclosure, the
cannabis extract
comprises at least one additional bioactive molecule isolated or derived from
cannabis. In some
embodiments, the at least one additional bioactive molecule comprises a
terpene or a flavonoid.
[00014] In some embodiments of the compositions of the disclosure, the at
least one
cannabinoid comprises A9 tetrahydrocannabinol (THC), cannabidiol (CBD),
tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabigerolic
acid
(CBGA), cannabichromenenic acid (CBCA), cannabigerovarinic acid (CBGVA),
tetrahydrocanabivarinic acid (THCVA), cannabidivarinic acid (CBDVA),
cannabichromevarinic acid (CBCVA), cannabinol (CBN), cannabigerol (CBG),
cannabichromene (CBC), cannabicyclol (CBL), cannabivarin (CBV),
tetrahydrocannabivarin
(THCV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin
(CBGV),
cannabigerol monomethylether (CBGM), cannabielsoin (CBE), cannabicitran (CBT),
or a
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combination thereof In some embodiments, the at least one cannabinoid
comprises a
combination of THC and CBD. In some embodiments, the at least one cannabinoid
comprises a
combination of THC, THCA, CBD and CBDA. In some embodiments, the at least one
cannabinoid comprises a THC metabolite. In some embodiments, the THC
metabolite
comprises 11-Hydroxy-A9-tetrahydrocannabinol (11-0H-THC).
[00015] In some embodiments of the compositions of the disclosure, the
composition
comprises about 2% to 20% cannabinoids. In some embodiments, the composition
comprises
about 5% to 15% cannabinoids. In some embodiments, the composition comprises
about 2% to
50% cannabinoids.
[00016] In some embodiments of the compositions of the disclosure, the
terpene comprises
myrcene, terpinolene,13-caryophyllene, selina-3 7(11)-diene, guaiol, 10-epi-y-
eudesmol, 13-
eudesmol, a-eudesmol, bulnesol, a-bisabolol, a-humulene, a-pinene, limonene,
linalool, or a
combination thereof
[00017] In some embodiments of the compositions of the disclosure, the
cannabis extract is
isolated from Cannabis sativa, Cannabis id/ca, Cannabis ruderahs, or a strain
or hybrid
thereof
[00018] In some embodiments of the compositions of the disclosure, the
lipid-based carrier
comprises marine oil. In some embodiments, the marine oil comprises fish oil.
In some
embodiments, the fish oil is isolated from Brevoortia, Clupea, Engrauhs,
Ethmidium, Sardina,
Sardinops, Scomber, Thunnus genera or a species of Gadidae . In some
embodiments, the
marine oil comprises krill oil. In some embodiments, the krill oil is isolated
from Euphausia
superba and/or Euphausia pacifica. In some embodiments, the krill oil
comprises
phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine
phospholipids. In
some embodiments, the concentration of phosphatidylcholine is at least 75% of
the total
phospholipid content. In some embodiments, the krill oil comprises
triglycerides and
phospholipids. In some embodiments, the ratio of triglycerides to
phospholipids is about 1:1. In
some embodiments, the ratio of triglycerides to phospholipids is about 1:1.3.
In some
embodiments, the ratio of triglycerides to phospholipids is about 1:1.7. In
some embodiments,
the ratio of triglycerides to phospholipids is about 1:3. In some embodiments,
the ratio of
triglycerides to phospholipids is about 1:4. In some embodiments, the ratio of
triglycerides to
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phospholipids is about 1:7. In some embodiments, the marine oil comprises
squid or seal oil. In
some embodiments, the marine oil comprises a mixture of fish oil and krill
oil. In some
embodiments, the lipid-based carrier has been treated to increase the level of
monoacylglycerides (MAG) in the lipid-based carrier.
[00019] In some embodiments of the compositions of the disclosure, the
composition
comprises MAG. In some embodiments, at least 4%, at least 5%, at least 6%, at
least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least
40%, at least 45% or at
least 50% of the total glycerides in the composition comprise
monoacylglycerides (MAG). In
some embodiments, at least 4% of the total glycerides in the composition
comprise MAG. In
some embodiments, at least 30% of the total glycerides in the composition
comprise MAG. In
some embodiments, at least 35% of the total glycerides in the composition
comprise MAG. In
some embodiments of the compositions of the disclosure, between about 4% to
50%, about
10% to 50%, about 20% to 50%, about 25% to 50%, about 30% to 50%, about 35% to
50%,
about 40% to 50%, about 45% to 50%, about 4% to 40%, about 10% to 40%, about
20% to
40%, about 30% to 40%, about 4% to 35%, about 10% to 35%, about 20% to 35%, or
about
30% to 35% of the total glycerides in the composition comprise MAG.
[00020] In some embodiments of the compositions of the disclosure, the
composition
comprises DAG. In some embodiments, at least 1%, at least 3%, at least 5%, at
least 7%, at
least 10%, at least 20%, at least 30%, at least 35% at least 40%, at least
45%, at least 47%, at
least 50%, at least 60%, or at least 70% of the glycerides in the composition
are
diacylglycerides (DAG).
[00021] In some embodiments, the composition comprises phospholipids. In
some
embodiments, the phospholipids are at least 20%, at least 25%, at least 35%,
at least 40%, at
least 50%, at least 60% or at least 70% of the lipids in the composition. In
some embodiments,
the composition comprises triglycerides. In some embodiments, the
phospholipids and the
triglycerides are present at about a 1:1 ratio.
[00022] In some embodiments of the compositions of the disclosure, the
composition
comprises an antioxidant. In some embodiments, the antioxidant comprises alpha
tocopherol, a
mixture of tocopherols, astaxanthin, or rosemary extract.
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[00023] In some embodiments of the compositions of the disclosure, the
composition
comprises a pharmaceutically acceptable carrier, diluent or excipient. In some
embodiments,
the pharmaceutically acceptable carrier comprises marine oil, fish oil, flax
seed oil, camelina
oil, evening primrose oil, black current oil, ahiflower seed oil, or a
combination thereof
[00024] In some embodiments of the compositions of the disclosure, the
composition is
formulated for oral administration. In some embodiments, the composition is
formulated as a
liquid, gel, softgel, powder, tablet, caplet, capsule, gelcap, food additive,
drop, beverage, pill,
lozenge, rinse, paste or gum.
[00025] In some embodiments of the compositions of the disclosure, the
composition is
formulated for transmucosal administration. In some embodiments, the
transmucosal
administration comprises buccal administration or intranasal administration.
[00026] In some embodiments of the compositions of the disclosure, the
bioavailability of
cannabinoids, terpenes, flavonoids or other bioactive molecules from the
cannabis extract is
greater than the bioavailability of the same molecules formulated in medium
chain triglyceride
(MCT). In some embodiments, the bioavailabity of cannabinoids in blood plasma
is increased
at least about 1.5X, at least about 2X, at least about. 2.25X or at least
about 2.5X compared to
the bioavailability cannabinoids formulated in MCT.
[00027] In some embodiments of the compositions of the disclosure, the
variability of
cannabinoid concentration in blood plasma following oral administration is
reduced compared
to the variability of cannabinoid concentration in blood plasma following oral
administration of
cannabinoids formulated in MCT.
[00028] In some embodiments of the compositions of the disclosure, the
effect on
cannabinoid bioavailability when administered in a fasting versus a fed state
is reduced
compared to the effect on cannabinoid bioavailability when administered in a
fasting versus a
fed state following oral administration of cannabinoids formulated in MCT.
[00029] The disclosure provides methods of making the compositions of the
disclosure,
comprising: (a) providing a cannabis extract; and (b) mixing the cannabis
extract with a lipid-
based carrier comprising omega-3 fatty acids and at least one of
monoacylglycerides,
diacylglycerides, triglycerides or phospholipids.
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[00030] The disclosure provides methods of making a composition comprising
a cannabis
extract, comprising (a) providing a cannabis extract; and (b) mixing the
cannabis extract with a
lipid-based carrier comprising omega-3 fatty acids, and at least one of
monoacylglycerides,
diacylglycerides, triglycerides or phospholipids.
[00031] In some embodiments of the methods of the disclosure, the cannabis
extract
comprises a liquid, a resin, a powder or an emulsion. Powders can be generated
by methods
such as spray drying, or by the addition of a plating agent or other additive
that can act as a
carrier. Spray drying is a method of producing a powder from a liquid or
slurry by rapidly
drying with hot gas. Exemplary plating agents include N-ZORBIT 2144 DG. In
some
embodiments, the cannabis extract is formulated as a powder or an emulsion and
comprises a
plating agent or carrier. Powders of desired particle size can be generated by
milling, which
subjects particles to mechanical stress, breaking the particles into smaller
sizes.
[00032] In some embodiments of the methods of the disclosure, the cannabis
extract has
been isolated from cannabis by lipid-based cold extraction, organic-solvent
based extraction,
supercritical fluid extraction, column chromatography, high performance liquid
chromatography (El:PLC) molecular distillation or a combination thereof In
some
embodiments, the cannabis extract is extracted from Cannabis sativa, Cannabis
id/ca,
Cannabis ruderalis, or a strain or hybrid thereof
[00033] In some embodiments of the methods of the disclosure, the liquid
comprises marine
oil, fish oil, flax seed oil, camelina oil, evening primrose oil, black
current oil, ahiflower seed
oil, or a combination thereof
[00034] In some embodiments of the methods of the disclosure, the lipid-
based carrier
comprises fish oil, krill oil, flax seed oil, a mixture or a derivative
thereof
[00035] In some embodiments of the methods of the disclosure, the lipid-
based carrier
comprises a marine oil. In some embodiments, the marine oil comprises fish
oil. In some
embodiments, the fish oil comprises MAG and DAG. In some embodiments, the
glycerides in
the fish oil comprise at least 4% MAG, at least 10% MAG, at least 20% MAG, at
least 25%
MAG, at least 30% MAG, at least 35% or at least 40% MAG. In some embodiments,
the
glycerides in the fish oil comprise at least 30% MAG or 35% MAG. In some
embodiments, the
glycerides in the fish oil comprises at least 10% DAG, at least 20% DAG, at
least 30% DAG, at
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least 40% DAG, at least 45% DAG or at least 50% DAG. In some embodiments, the
glycerides
in the fish oil comprise at least 40% DAG or 45% DAG. In some embodiments, the
fish oil
comprises MAG:DAG at a ratio of about 0.8:1.
[00036] In some embodiments of the methods of the disclosure, the lipid-
based carrier
comprises a marine oil. In some embodiments, the marine oil comprises krill
oil. In some
embodiments, the krill oil comprises phospholipids and triglycerides. In some
embodiments,
the phospholipids are at least 20%, at least 25%, at least 35%, at least 40%,
50%, at least 60%
or at least 70% of the lipids in the krill oil. In some embodiments, the
phospholipids and
triglycerides are present at about a 1:1 ratio of triglycerides:phospholipids
in the krill oil. In
some embodiments, the ratio of triglycerides to phospholipids is about 1:1.3.
In some
embodiments, the ratio of triglycerides to phospholipids is about 1:1.7. In
some embodiments,
the ratio of triglycerides to phospholipids is about 1:3. In some embodiments,
the ratio of
triglycerides to phospholipids is about 1:4. In some embodiments, the ratio of
triglycerides to
phospholipids is about 1:7.
[00037] In some embodiments of the methods of the disclosure, the marine
oil comprises a
mixture of fish and krill oil.
[00038] In some embodiments of the methods of the disclosure, the cannabis
extract is
mixed with the lipid-based carrier at a ratio of about 1:7, about 1:8, about
1:9, about 1:9.5,
about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about
1:16, about 1:17,
about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about
1:24 or about 1:25
cannabis extract to lipid-based carrier.
[00039] Unless otherwise defined, all technical and scientific terms used
herein have the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention pertains. Although methods and materials similar or equivalent to
those described
herein can be used in the practice of the present invention, suitable methods
and materials are
described below. All publications, patent applications, patents, and other
references mentioned
herein are expressly incorporated by reference in their entirety. In cases of
conflict, the present
specification, including definitions, will control. In addition, the
materials, methods, and
examples described herein are illustrative only and are not intended to be
limiting.
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[00040] Other features and advantages of the invention will be apparent
from and
encompassed by the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[00041] FIG. 1 is a plot showing the plasma concentration-time profile of
cannabidiol
(CBD) from cannabis extract in different carrier oils following oral (gavage)
administration to
rats.
[00042] FIGS. 2A-2D are plots showing the pharmacokinetic parameters of
cannabidiol
(CBD) absorption from cannabis extract in different carrier oils following
oral (gavage)
administration to rats. FIG. 2A shows area under the curve (AUC). FIG. 2B
shows maximum
concentration (Cmax). FIG. 2C shows absolute bioavailability. FIG. 2D shows
relative
bioavailability. Dark gray bars are relative to medium chain triglyceride
(MCT), while light
gray bars are relative to TG-03.
[00043] FIG. 3 is a plot showing the plasma concentration-time profile of
tetrahydrocannabinol (THC) from cannabis extract in different carrier oils
following oral
(gavage) administration to rats.
[00044] FIGS. 4A-4D are plots showing the pharmacokinetic parameters of
tetrahydrocannabinol (THC) absorption from cannabis extract in different
carrier oils following
oral (gavage) administration to rats. FIG. 4A shows AUC. FIG. 4B shows Cmax.
FIG. 4C
shows absolute bioavailability. FIG. 4D shows relative bioavailability. Dark
gray bars are
relative to medium chain triglyceride (MCT), while light gray bars are
relative to TG-03.
[00045] FIG. 5 is a plot showing the plasma concentration-time profile of
cannabidiol
(CBD) from cannabis extract in different carrier oils following oral (gavage)
administration to
rats
[00046] FIGS. 6A-6D are plots showing the pharmacokinetic parameters of
cannabidiol
(CBD) absorption from cannabis extract in different carrier oils following
oral (gavage)
administration to rats. FIG. 6A shows AUC. FIG. 6B shows Cmax. FIG. 6C shows
absolute
bioavailability. FIG. 6D shows relative bioavailability. Dark gray bars are
relative to medium
chain triglyceride (MCT), while light gray bars are relative to TG-03.
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[00047] FIG. 7 is a plot showing the plasma concentration-time profile of
tetrahydrocannabinol (THC) from cannabis extract in different carrier oils
following oral
(gavage) administration to rats.
[00048] FIGS. 8A-8D are plots showing the pharmacokinetic parameters of
tetrahydrocannabinol (THC) absorption from cannabis extract in different
carrier oils following
oral (gavage) administration to rats. FIG. 8A shows AUC. FIG. 8B shows Cmax.
FIG. 8C
shows absolute bioavailability. FIG. 8D shows relative bioavailability. Dark
gray bars are
relative to medium chain triglyceride (MCT), while light gray bars are
relative to TG-03.
DETAILED DESCRIPTION OF THE INVENTION
[00049] Oral absorption of lipophilic molecules, such as cannabinoids, is
an ongoing
challenge, as uptake of compounds from the gastrointestinal tract favors those
with hydrophilic
properties. There are many lipophilic drugs (e.g., clofazimine as Lamprene;
saquinavir as
Invirase; and efavirenz as Sustiva), as well as many lipophilic nutrients
(e.g., vitamin D, lutein,
and coenzyme Q10), that are taken orally and are poorly transported across the
intestinal
epithelium and/or are first metabolized by the liver, and hence this route of
administration does
not facilitate optimal concentrations in the systemic circulation.
[00050] To overcome the poor absorption of lipophilic drugs/nutrients,
lipid-based drug
delivery systems (LBDDS) are commonly used (Griffin, 2012). Without LBDDS,
lipophilic
compounds coalesce in the stomach upon ingestion, as the gastric fluid is
water-based.
However, if the same compounds are formulated with excipients that act as
emulsifiers, it
allows the lipids to disperse into small micelles, thereby improving
solubility and permeability,
and increasing absorption. The composition and concentration of the excipients
used (mono-
and diglycerides, phospholipids, medium chain triglycerides, etc.) will result
in micelles of
varying sizes, ranging in the scale of nanometers to micrometers.
[00051] In the nutrition industry, pre-clinical and human clinical trials
(Maki et al., 2009;
Cruz-Hernandez et al., 2016; Ahn et al., 2018) have involved the study of the
effects of
combinations of lipophilic molecules and omega-3 fatty acids with a
monoacylglyceride or
phospholipid backbone, with a reported increase in absorption of said
molecules versus
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administration via standard triglyceride omega-3 fatty acids. The exact
mechanism of action is
not fully elucidated. However, without wishing to be bound by theory, it is
thought that these
monoglycerol or phospholipid backbone molecules are acting in a similar
fashion to a self-
microemulsifying drug delivery system (SMEDDS) for the lipophilic compounds.
[00052] In order to assess the ability of monoacylglyceride- and
phospholipid-rich carriers to
increase the oral absorption of cannabinoids (CBs) from a cannabis plant
extract through the
gastrointestinal barrier, the inventors have evaluated the utility of
different carrier oils,
including a monoacylglyceride-rich fish oil (MAG-03), krill oil (PL-03) and
flax seed oil (TG-
03). These carrier oils are rich in long-chain omega-3 fatty acids complexed
to
monoacylglycerides (MAG-03), phospholipids (PL-03), or triglycerides (flax
seed oil, TG-
03). A pre-clinical study was therefore designed to compare the relative and
absolute
bioavailability of a cannabis plant extract (1:1 THC:CBD ratio) administered
as a blend of each
of these omega-3 fatty acid oil preparations (i.e., MAG-03/CBs, PL-03/CBs, and
TG-03/CBs)
compared to a commonly-used carrier oil, medium chain triglycerides (MCT, from
coconut
oil)/CBs. An intravenous dose of lipid-free CB extract was given to act as a
baseline and for
use in the calculation of absolute bioavailability (fabs).
[00053] Cannabis extracts formulated with the carrier oils of the invention
were determined
to have improved (i.e. increased) bioavailability, as well as improved (i.e.,
reduced) variability
in bioavailability, and thus improved predictability of the effect when dosing
cannabinoids
compared to formulations using other oils, such as medium chain triglycerides.
Lipid-Based Carriers
[00054] The disclosure provides lipid-based carriers that can be mixed with
the cannabis
extracts described herein to produce cannabis extract compositions with
increased
bioavailability when compared to cannabis extracts formulated in other lipid-
based carriers,
such as medium chain triglycerides (MCT).
[00055] In some embodiments, the lipid-based carrier comprises omega-3
fatty acids. In
some embodiments, the lipid based carrier comprises monoacylglycerides,
diacylglycerides and
phospholipids. In some embodiments, the omega-3 fatty acids are omega-3
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monoacylglycerides, omega-3 diacylglycerides, omega-3 phospholipids or a
combination
thereof
[00056] As used herein, "glycerides", also known as" acyglycerols", refers
to a class of
molecules where esters are formed between a glycerol and a fatty acid. An
"acylglyceride
linkage" refers to the covalent bond between the organic acid group, such as a
fatty acid, and
one of the three hydroxyl groups of the glycerol, for example via an ester
linkage.
[00057] As used herein, "monoacylglycerides", or "MAG", sometimes also
referred to as
"monoglycerides" or "monoacylglycerols" are a class of glycerides that are
composed of a
molecule of glycerol linked to a fatty acid via an ester bond. Glycerol
contains both primary
and secondary alcohol groups. Therefore, two different types of monoglycerides
may be
formed: 1-monoacylglycerols where the fatty acid is attached to a primary
alcohol, and 2-
monoacylglycerols where the fatty acid is attached to the secondary alcohol.
[00058] "Diacylglycerides", or "DAG", sometimes referred to as
"diglyceride" or
"diacylglycol", refers to a glyceride composed of two fatty acids covalently
linked to a glycerol
molecule through ester linkages. Two possible forms exist: 1,2-diacylglycerols
and 1,3-
diacylglycerols.
[00059] "Triglycerides", sometimes referred to as "TG", "TAG",
"triacylglycerol" or
"triacylglyceride" are molecules comprising a glycerol linked to three fatty
acids via ester
linkages.
[00060] The term "fatty acid(s)" as used herein refers to long-chain
aliphatic acids (alkanoic
acids) of varying chain lengths, from about C12 to C22 (although both longer
and shorter
chain-length acids are known). For example, the predominant chain lengths are
about C16 to
about C22. The structure of a fatty acid is represented by a simple notation
system of "XY",
where X is the total number of carbon (C) atoms and Y is the number of double
bonds.
[00061] Generally, fatty acids are classified as saturated or unsaturated.
The term "saturated
fatty acids" refers to those fatty acids that have no "double bonds" between
their carbon
backbone. In contrast, "unsaturated fatty acids" are cis- or trans-isomers
that have "double
bonds" along their carbon backbones. "Monounsaturated fatty acids" have only
one "double
bond" along the carbon backbone (e.g., usually between the 9th and 10th carbon
atom as for
palmitoleic acid (16:1) and oleic acid (18:1)), while "polyunsaturated fatty
acids" (or "PUFAs")
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have at least two double bonds along the carbon backbone (e.g., between the
9th and 10th, and
12th and 13th carbon atoms for linoleic acid (18:2) and between the 9th and
10th, 12th and
13th, and 15th and 16th for alpha-linolenic acid (18:3)).
[00062] PUFAs can be classified into two major families (depending on the
position (n) of
the first double bond nearest the methyl end of the fatty acid carbon chain).
Thus, the "omega-6
fatty acids" (omega-6 or n-6) have the first unsaturated double bond six
carbon atoms from the
omega (methyl) end of the molecule and additionally have a total of two or
more double bonds,
with each subsequent unsaturation occurring 3 additional carbon atoms toward
the carboxyl end
of the molecule. In contrast, the "omega-3 fatty acids" (omega-3 or n-3) have
the first
unsaturated double bond three carbon atoms away from the omega end of the
molecule and
additionally have a total of three or more double bonds, with each subsequent
unsaturation
occurring 3 additional carbon atoms toward the carboxyl end of the molecule.
[00063] As used herein, "omega-3 fatty acids", also called "(p-3 fatty
acids" or "n-3 fatty
acids" refers to polyunsaturated fatty acids (PUFAs) that are characterized by
the presence of a
double bond three atoms away from the terminal methyl group of the fatty acid.
Exemplary
omega-3 fatty acids include a-linolenic acid (ALA) found in plant oils, and
eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA), both commonly found in marine oils.
Common
sources of plant oils containing ALA include walnut, edible seeds, clary sage
seed oil, algal oil,
flax seed oil, Sacha Inchi oil, Echium oil, and hemp seed oil. Common sources
of the omega-3
fatty acids, EPA and DHA, include fish, fish oils, eggs from chickens fed EPA
and DHA, algal
oil, squid oil, and krill oil.
[00064] A "lipid" is a molecule that is soluble in nonpolar solvents.
Lipids include fats, fatty
acids and their derivatives, as well as sterol-containing metabolites such as
cholesterols and
waxes.
[00065] A "phospholipid" refers to a class of lipid comprising two
hydrophobic fatty acid
tails and a hydrophilic head comprising a phosphate group, which can be joined
via a glycerol
molecule. The phosphate groups of the head can be modified with organic
molecules such as
choline, ethanolamine or serine.
[00066] An "omega-3-containing phospholipid" is a phospholipid where one or
both of the
fatty acid tails of the phospholipid is an omega-3 fatty acid.
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[00067] Exemplary monoacylglycerides include compounds of formulas (I),
(II), (III), and
(IV):
x(-3c,'112
X2,
RI
0
X)e2
III
X2,
RI
0
X)e2
X2,
RI
IV
R1 \ x2
XR2
0
wherein X1 is 0, NH, or S; X2 is 0, NH, or S; X3 is 0, NH, or S; R1 and R2
each
independently represents ¨H, ¨C(0)NH2, ¨S(0)NH2, ¨S(0)2NH2, ¨C1-C22
(oxy)alkyl,
¨C1-C22 alkyl, ¨C1-C22 (hydroxy)alkyl, ¨C1-C22 (amino)alkyl, ¨C1-C22
(halo)alkyl, ¨
C3-C22 alkenyl, ¨C3-C22 alkynyl, ¨(C3-C7) cycloalkyl unsubstituted or
substituted with at
least one substituent chosen from C1-C22 alkyl, ¨C2-C22 alkenyl, and ¨C2-C22
alkynyl, ¨
C6-C12 aryl, ¨C7-C22 (aryl)alkyl, ¨C8-C22 (aryl)alkenyl, ¨C8-C22
(aryl)alkynyl, three- to
seven-membered non-aromatic heterocycle unsubstituted or substituted with at
least one
substituent chosen from ¨C1-C22 alkyl, ¨C2-C22 alkenyl, and ¨C2-C22 alkynyl,
five- to
seven-membered aromatic heterocycle unsubstituted or substituted with at least
one substituent
chosen from ¨C1-C22 alkyl, ¨C2-C22 alkenyl, and ¨C2-C22 alkynyl, ¨(CH2)n amino
acid
wherein the amino acid is connected through its alpha carbon atom, ¨(CH2)n
peptide wherein
the peptide is connected through the alpha carbon atom of one of its amino
acids, ¨CH2OR5,
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¨C(0)R5, ¨C(0)0R5, ¨C(0)NR5, ¨P(0)(0R5)2, ¨S(0)2NHR5, ¨SOR5, ¨S(0)2R5, -
arylP(0)(0R5)2, a sugar, or a sugar phosphate or R1 and R2 are joined together
so as to form a
five- to seven-membered non-aromatic heterocycle unsubstituted or substituted
with at least
one substituent chosen from ¨C1-C22 alkyl, ¨C2-C22 alkenyl, and ¨C2-C22
alkynyl, a
phosphate, sulfate carbonyl group, or a thiocarbonyl imine; R5 is ¨H, ¨C1-C22
alkyl, ¨(C3-
C7) cycloalkyl, ¨C1-C22 (halo)alkyl, ¨C6-C12 aryl, ¨C2-C22 alkenyl, ¨C2-C22
alkynyl,
¨C7-C22 (aryl)alkyl, ¨C8-C22 (aryl)alkenyl, ¨C8-C22 (aryl)alkynyl, ¨C1-C22
(hydroxy)alkyl, ¨C1-C22 alkoxy, ¨C1-C22 (amino)alkyl, a ¨(C3-C7) cycloalkyl
unsubstituted or substituted with at least one substituent chosen from ¨C1-C22
alkyl, ¨C2-
C22 alkenyl, and ¨C2-C22 alkynyl, a three- to seven-membered non-aromatic
heterocycle
unsubstituted or substituted at least one substituent chosen from ¨C1-C22
alkyl, ¨C2-C22
alkenyl, and ¨C2-C22 alkynyl, a three- to seven-membered aromatic heterocycle
unsubstituted
or substituted with at least one substituent chosen from ¨C1-C22 alkyl, ¨C2-
C22 alkenyl,
and ¨C2-C22 alkynyl, a ¨(CH2)n amino acid wherein the amino acid is connected
to the
compound through its alpha carbon atom, a ¨(CH2)n peptide wherein the peptide
is connected
to the compound through the alpha carbon atom of one of its amino acids, a
sugar or a sugar
phosphate; and n is an integer having a value of 0, 1, 2, 3, or 4, and
pharmaceutically
acceptable salts thereof
[00068] Further exemplary monoacylglycerides include compounds of formulas
(V), (VI),
(VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV) or (XV):
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PCT/CA2020/051081
V
0
Xr."-------"\
X3
X2,._
R,
R3
VI
0
XrN''--------\
x3
- - X2 ----_(
R4
R3
VII
0
Xr'N------"A
X3
X2__..,
R4
R3
VIII
R3
X-.---.' R4
X1,,,,,LIX3
0
IX
0
Xr.'=--------\
0
ç
/
HO
X
0
Xr'"--------\
0
0 /
--13
...k.
/ 0
HO
x ._.,
CA 03149652 2022-02-02
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XI
IIOH
0 \
0
0
XII
0
0
XIII
0
xo
XIV
0
'.70
0
XV
0
II 0
0-- \o
0
Xi is 0, NH, or S; X2 is 0, NH, or S; X3 is 0, NH, or S; R3 and R4 each
independently
represents ¨H, ¨C(0)NH2, ¨S(0)NH2, ¨S(0)2NH2, ¨C1-C22 (oxy)alkyl, ¨C1-C22
alkyl,
¨C1-C22 (hydroxy)alkyl, ¨C1-C22 (amino)alkyl, ¨C1-C22 (halo)alkyl, ¨C3-C22
alkenyl,
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¨C3-C22 alkynyl, ¨(C3-C7) cycloalkyl unsubstituted or substituted with at
least one
substituent chosen from C1-C22 alkyl, ¨C2-C22 alkenyl, and ¨C2-C22 alkynyl,
¨C6-C12
aryl, ¨C7-C22 (aryl)alkyl, ¨C8-C22 (aryl)alkenyl, ¨C8-C22 (aryl)alkynyl, three-
to seven-
membered non-aromatic heterocycle unsubstituted or substituted with at least
one substituent
chosen from ¨C1-C22 alkyl, ¨C2-C22 alkenyl, and ¨C2-C22 alkynyl, five- to
seven-
membered aromatic heterocycle unsubstituted or substituted with at least one
substituent
chosen from ¨C1-C22 alkyl, ¨C2-C22 alkenyl, and ¨C2-C22 alkynyl, ¨(CH2)n amino
acid
wherein the amino acid is connected through its alpha carbon atom, ¨(CH2)n
peptide wherein
the peptide is connected through the alpha carbon atom of one of its amino
acids, ¨CH2OR5,
¨C(0)R4, ¨C(0)0R4, ¨C(0)NR4, ¨P(0)(0R5)2, ¨S(0)2NHR5, ¨SOR5, ¨S(0)2R5, -
arylP(0)(0R5)2, a sugar, or a sugar phosphate, or R3 and R4 are joined
together so as to form a
five- to seven-membered non-aromatic heterocycle unsubstituted or substituted
with at least
one substituent chosen from ¨C1-C22 alkyl, ¨C2-C22 alkenyl, and ¨C2-C22
alkynyl, a
phosphate, sulfate carbonyl group, or a thiocarbonyl imine; R5 is ¨H, ¨C1-C22
alkyl, ¨(C3-
C7) cycloalkyl, ¨C1-C22 (halo)alkyl, ¨C6-C12 aryl, ¨C2-C22 alkenyl, ¨C2-C22
alkynyl,
¨C7-C22 (aryl)alkyl, ¨C8-C22 (aryl)alkenyl, ¨C8-C22 (aryl)alkynyl, ¨C1-C22
(hydroxy)alkyl, ¨C1-C22 alkoxy, ¨C1-C22 (amino)alkyl, a ¨(C3-C7) cycloalkyl
unsubstituted or substituted with at least one substituent chosen from ¨C1-C22
alkyl, ¨C2-
C22 alkenyl, and ¨C2-C22 alkynyl, a three- to seven-membered non-aromatic
heterocycle
unsubstituted or substituted at least one substituent chosen from ¨C1-C22
alkyl, ¨C2-C22
alkenyl, and ¨C2-C22 alkynyl, a three- to seven-membered aromatic heterocycle
unsubstituted
or substituted with at least one substituent chosen from ¨C1-C22 alkyl, ¨C2-
C22 alkenyl,
and ¨C2-C22 alkynyl, a ¨(CH2)n amino acid wherein the amino acid is connected
to the
compound through its alpha carbon atom, a ¨(CH2)n peptide wherein the peptide
is connected
to the compound through the alpha carbon atom of one of its amino acids, a
sugar or a sugar
phosphate; and n is an integer having a value of 0, 1, 2, 3, or 4; and
pharmaceutically
acceptable salts thereof
[00069] The sugar can be chosen from 5-carbon sugars and 6-carbon sugars.
Non-limiting
examples of 5-carbon sugar include ribose, arabinose, xylose, and lyxose. Non-
limiting
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examples of 6-carbon sugar include glucose, galactose, mannose, allose,
gulose, idose, talose,
and altrose.
[00070] The sugar phosphate can be chosen from monosaccharides (such as
mannose-6-
phosphate, glucose-6-phosphate, galactose-6-phosphate, mannose-l-phosphate,
glucose-1-
phosphate and galactose-1-phosphate), disaccharides (such as 6-0-phosphoryl-a-
D-
mannopyranosyl-(1-2)-D-mannopyranose, 6-0-phosphoryl-a-D-mannopyranosyl-(1-3)-
mannopyranose, 6-0-phosphoryl-a-D-mannopyranosyl-(1-6)-D-mannopyranose),
trisaccharides (such as 6-0-phosphoryl-a-D-mannopyranosyl-(1-2)-D-
mannopyranosyl-(1-2)-
D-mannopyranose), and higher linear or branched oligosaccharides (such as
pentamannose-6-
phosphate).
[00071] The amino acid can be chosen from alanine, arginine, asparagine,
aspartic acid,
cysteine, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine,
methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
[00072] The peptide can be chosen from any possible combination of the
amino acids
previously described.
[00073] The term "aryl" as used herein refers to a cyclic or polycyclic
aromatic ring. For
example, the aryl group can be phenyl or napthyl.
[00074] The expression "aromatic heterocycle" as used herein refers to an
aromatic cyclic or
fused polycyclic ring system having at least one heteroatom selected from the
group consisting
of N, 0, S and P. Non-limitative examples include heteroaryl groups are furyl,
thienyl, pyridyl,
quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl,
tetrazolyl, imidazolyl,
pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl,
benzoxazolyl,
pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl,
isoxazolyl,
isothiazolyl, purinyl, quinazolinyl, and so on.
[00075] The expression "non-aromatic heterocycle" includes non-aromatic
rings or ring
systems that contain at least one ring having at least one hetero atom (such
as nitrogen, oxygen,
sulfur or phosphorus). This term includes, in a non-limitative manner all of
the fully saturated
and partially unsaturated derivatives of the above mentioned aromatic
heterocycles groups.
Examples of non-aromatic heterocycle groups include, in a non-limitative
manner, pyrrolidinyl,
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tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl,
thiazolidinyl,
isothiazolidinyl, and imidazolidinyl.
[00076] The expression "pharmaceutically acceptable acid addition salt" as
used herein
means any non-toxic organic or inorganic salt of any compounds of the present
disclosure, or
any of its intermediates. Illustrative inorganic acids which form suitable
salts include
hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal
salts such as sodium
monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative
organic acids that
form suitable salts include mono-, di-, and tricarboxylic acids such as
glycolic, lactic, pyruvic,
malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic,
maleic, benzoic,
phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as
p-toluene sulfonic
and methanesulfonic acids. Either the mono or di-acid salts can be formed, and
such salts may
exist in either a hydrated, solvated or substantially anhydrous form. In
general, the acid
addition salts of the compounds of the present disclosure are more soluble in
water and various
hydrophilic organic solvents, and generally demonstrate higher melting points
in comparison to
their free base forms. The selection of the appropriate salt will be known to
one skilled in the
art. Other non-pharmaceutically acceptable salts, e.g. oxalates, may be used,
for example, in the
isolation of the compounds of the present disclosure, for laboratory use, or
for subsequent
conversion to a pharmaceutically acceptable acid addition salt. In embodiments
of the present
disclosure, the pharmaceutically acceptable acid addition salt is the
hydrochloride salt.
[00077] The term "pharmaceutically acceptable basic addition salt" as used
herein means
any non-toxic organic or inorganic base addition salt of any acid compound of
the disclosure,
or any of its intermediates. Acidic compounds of the disclosure that may form
a basic addition
salt include, for example, where R is CO2H. Illustrative inorganic bases which
form suitable
salts include lithium, sodium, potassium, calcium, magnesium or barium
hydroxide. Illustrative
organic bases which form suitable salts include aliphatic, alicyclic or
aromatic organic amines
such as methylamine, trimethylamine and picoline or ammonia. The selection of
the
appropriate salt will be known to a person skilled in the art. Other non-
pharmaceutically
acceptable basic addition salts, may be used, for example, in the isolation of
the compounds of
the disclosure, for laboratory use, or for subsequent conversion to a
pharmaceutically
acceptable acid addition salt.
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[00078] In some embodiments, the lipid-based carrier comprises a vegetable
or seed oil
(such as flax seed oil, pumpkin seed oil, canola oil, soybean oil, or walnut
oil), fish oil (such as
cod liver oil, salmon oil, tuna oil, shark oil, pelagic fishes oil, mackerel
oil, or sardine oil), seal
oil, microalgae oil, krill oil, crustacean oil (for example shrimp oil),
mussel oil (for example
green lipped mussel oil), squid oil, or mixtures thereof In some embodiments,
the lipid-based
carrier comprises an oil that has been processed to increase the percentage of
MAG, DAG,
triglycerides or phospholipids, or a combination thereof, in the oil.
[00079] In some embodiments, the lipid-based carrier comprises a vegetable
or seed oil
(such as flax seed oil, pumpkin seed oil, canola oil, soybean oil, walnut oil,
camelina oil,
evening primrose oil, black current oil, ahiflower seed oil), a marine oil
(such as algae oil, seal
oil, krill oil, crustacean oil, or fish oil, for example cod liver oil, salmon
oil, tuna oil, shark oil,
pelagic fishes oil, mackerel oil, sardine oil, or anchovy oil), or an
hydrolysate. In some
embodiments, the lipid-based carrier comprises a vegetable or seed oil, or a
marine oil, that has
been processed to increase the percentage of MAG and/or DAG in the oil.
[00080] In some embodiments, the marine oil comprises krill oil. In some
embodiments, the
krill oil is isolated from Euphausia superba and/or Euphausia pacifica. In
some embodiments,
the krill oil comprises phosphatidylcholine, phosphatidylinositol, and
phosphatidylethanolamine phospholipids. In some embodiments, the concentration
of
phosphatidylcholine is at least 75% of the total phospholipid content. In some
embodiments,
the concentration of phosphatidylcholine is at least 60%, at least 70%, at
least 75%, at least
80% or at least 85% of the total phospholipid content. In some embodiments,
the phospholipids
are at least 20%, at least 25%, at least 35%, at least 40%, 50%, at least 60%
or at least 70% of
the lipids in the krill oil. In some embodiments, the krill oil comprises
phospholipids and
triglycerides. In some embodiments, the ratio of phospholipids to
triglycerides is about 1:1. In
some embodiments, the krill oil has been processed to increase the percentage
of phospholipids
and/or triglycerides in the krill oil.
[00081] In some embodiments, the lipid-based carrier comprises a marine
oil. In some
embodiments, the marine oil comprises fish oil isolated from Brevoortia,
Clupea, Engrauhs,
Ethmidium, Sardina, Sardinops, Scomber, Thunnus genera or a species of Gadidae
. In some
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embodiments, the marine oil comprises squid or seal oil. In some embodiments,
the marine oil
has been processed to increase the percentage of MAG and/or DAG in the marine
oil.
[00082] In some embodiments, the marine oil comprises a mixture of fish oil
and krill oil.
[00083] In some embodiments, the lipid-based carrier comprises at least one
monoacylglyceride (MAG). In some embodiments, the at least one MAG present in
the lipid-
based carrier can be a fatty acid or a derivative thereof For example, the MAG
is a C1-C6 ester
(C1-C6 being the amount of carbon atoms in the "alcohol" portion of the ester)
of a fatty acid
such as an ethyl ester) or a pharmaceutically acceptable salt thereof In some
embodiments, the
at least one MAG present in the lipid-based carrier can be an omega-3
monoacylglyceride.
[00084] In some embodiments, at least 4%, at least 5%, at least 6%, at
least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45% or at least
50% of the total glycerides in the lipid-based carrier comprise
monoacylglycerides (MAG).
[00085] In some embodiments, at least 30% of the total glycerides in the
lipid-based carrier
comprise MAG. In some embodiments, at least 35% of the total glycerides in the
lipid-based
carrier comprise MAG. In some embodiments, at least 40% of the total
glycerides in the lipid-
based carrier comprise MAG. In some embodiments, at least 45% of the total
glycerides in the
lipid-based carrier comprise MAG. In some embodiments, at least 50% of the
total glycerides
in the lipid-based carrier comprise MAG.
[00086] In some embodiments, between about 4% to 70%, about 10% to 70%,
about 20% to
70%, about 25% to 70%, about 30% to 70%, about 35% to 70%, about 40% to 70%,
about 45%
to 70%, about 50% to 70%, about 60% to 70%, about 4% to 60%, about 10% to 60%,
about
20% to 60%, about 25% to 60%, about 30% to 60%, about 35% to 60%, about 40% to
60%,
about 45% to 60%, about 50% to 60%, about 4% to 50%, about 10% to 50%, about
20% to
50%, about 25% to 50%, about 30% to 50%, about 35% to 50%, about 40% to 50%,
about 45%
to 50%, about 4% to 40%, about 10% to 40%, about 20% to 40%, about 30% to 40%,
about
35% to 40%, about 4% to 35%, about 10% to 35%, about 20% to 35%, about 30% to
35%,
about 4% to 30%, about 10% to 30% or about 20% to 30%, of the total glycerides
in the lipid-
based carrier comprise MAG.
[00087] In some embodiments, the lipid-based carrier comprises at least one
DAG.
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[00088] In some embodiments, at least 1%, at least 3%, at least 5%, at
least 7%, at least
10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 47%, at least
50%, at least 60%, at least 70%, at least 80% or at least 85% of the
glycerides in the lipid-
based carrier comprise DAG.
[00089] In some embodiments, at least 40% of the glycerides in the lipid-
based carrier
comprise DAG.
[00090] In some embodiments, the ratio of MAG:DAG in the lipid-based
carrier is about
0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about
1.1:1, about 1.2:1,
about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1,
about 1.9:1, about
2:1, about 2.1:1, about 2.2:1, about 2.3:1, about 2.4:1, about 2.5:1 or about
3:1.
[00091] Methods of measuring and determining type and concentration of
glycerides will be
readily apparent to the person of ordinary skill in the art. Exemplary methods
include liquid
chromatography, supercritical chromatography and high temperature gas
chromatography.
[00092] In some embodiments, the lipid-based carrier comprises an oil that
has been treated
to increase the percentage of MAG, DAG, triglycerides and/or phospholipids in
the oil. For
example, the lipid-based carrier comprises a vegetable or seed oil (such as
flax seed oil,
pumpkin seed oil, canola oil, soybean oil, walnut oil, camelina oil, evening
primrose oil, black
current oil, ahiflower seed oil), a marine oil (such as algae oil, seal oil,
krill oil, crustacean oil,
or fish oil (for example cod liver oil, salmon oil, tuna oil, shark oil,
pelagic fishes oil, mackerel
oil, sardine oil, or anchovy oil)) that has been treated to increase the
percentage of MAG, DAG,
triglycerides or phospholipids in the oil.
[00093] Methods of increasing the percentage of MAG in suitable lipid-based
carriers will
be readily apparent to the person of ordinary skill in the art. Exemplary
methods of generating
suitable lipid-based carriers with enriched MAG content are described in US
patent Nos.
8,119,690; 8,329,747; 8,816,110; 9,233,915; 8,222,295; 8,198,324; 9,101,563;
8,722,737;
9,480,660 and 9,925,165, the contents of each of which are incorporated by
reference in their
entireties herein. For example, a suitable starting lipid-based carrier
containing DAG and/and
triacyglycerides (TAG) can be subjected to hydrolysis of the DAG and/or TAG by
lipases such
as diacylglycerol lipase (DAG) or lipoprotein lipase (TAG). Suitable lipases
include, but are
not limited to Candida antartica lipase. Suitable starting lipid-based
carriers include, but are
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not limited to vegetable or seed oils (such as flax seed oil, pumpkin seed
oil, canola oil,
soybean oil, walnut oil, camelina oil, evening primrose oil, black current
oil, ahiflower seed
oil), marine oils (such as algae oil, seal oil, krill oil, crustacean oil, or
fish oil (for example cod
liver oil, salmon oil, tuna oil, shark oil, pelagic fishes oil, mackerel oil,
sardine oil, or anchovy
oil)). In some embodiments, the starting lipid-based carrier that is enriched
for MAG comprises
a marine oil. In some embodiments, the starting lipid-based carrier that is
enriched for MAG
comprises krill oil. In some embodiments, the starting lipid-based carrier
that is enriched for
MAG comprises fish oil, for example fish oil isolated from Brevoortia, Clupea,
Engrauhs,
Ethmidium, Sardina, Sardinops, Scomber, Thunnus genera or a species of
Gadidae. In some
embodiments, the starting lipid-based carrier that is enriched for MAG
comprises flax seed oil.
Bioavailability
[00094] The lipid-based carriers described herein increase the
bioavailability of
cannabinoids such THC and CBD compared to the bioavailability of cannabinoids
administered
using other carriers. Additional advantages of the cannabinoid compositions
described herein
include reduced variability in bioavailability when administered to a subject
as a result of
lessened influence by fatty food intake, and therefore increased
predictability in dosing subjects
with cannabinoids.
[00095] Bioavailability" refers to the proportion of a drug or other
substance that enters the
circulatory system when administered to a subject, and is so able to have an
active effect.
Methods of measuring bioavailability include administering the cannabis
extract compositions
described herein to a subject, and then measuring plasma concentrations of
cannabis extract
compounds using methods known in the art (e.g., gas chromatography or mass
spectrometry).
An exemplary method of measuring cannabinoids comprises microflow liquid
chromatography,
for example using a UPLC HSS-T3 column (100 mm * lmm, 1.8 [tm, equipped with a
0.2 [tm
fitted pre-filter).
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Cannabis Extracts
[00096] The disclosure provides compositions comprising a cannabis extract
comprising at
least one cannabinoid, and a lipid-based carrier. In some embodiments, the
cannabis extract
comprises at least one additional bioactive molecule isolated or derived from
cannabis, such as
a terpene, flavonoid, or other bioactive molecule.
Cannabis
[00097] Cannabis is a genus of plants that include three species, Cannabis
sativa, Cannabis
indica, and Cannabis ruderalis. More generally, cannabis also is categorized
as either
marijuana or hemp based on the natural amount of A9-tetrahydrocannabinol (THC)
present in
the plant material, with marijuana being high in THC and hemp having
negligible to no amount
of THC. This genus has long been used for its hemp fiber material, as well as
milk, seeds and
oils, for medicinal purposes, and for recreational use. Cannabis species
contain a highly
complex mixture of compounds, and up to 568 unique molecules have been
identified to date
(Lewis et al., 2017), any one of which is potentially bioactive in humans.
Exemplary bioactive
molecules in cannabis comprise cannabinoids, terpenes and flavonoids.
[00098] A variety of strains and hybrids of Cannabis will be known to the
person of
ordinary skill in the art, all of which can be used as starting material to
produce the cannabis
extracts used in the compositions and methods described herein. Different
Cannabis strains
produce different amounts of various cannabinoids and/or terpenes, and choice
of Cannabis
strain(s) or hybrid(s) can contribute to the cannabinoid and/or terpene
composition of the
cannabis extracts produced using the methods described herein. The person of
ordinary skill in
the art will be able to select the starting Cannabis strain or hybrid most
suited to the desired
cannabinoid and/or terpene composition of the cannabis extract. For example,
high cannabidiol
(CBD) strains include Charlotte's Web, Cannatonic, AC/DC, Harlequin, Ringo's
Gift, Harle-
Tsu, Nebula and Sour Tsunami. Exemplary high A9-tetrahydrocannabinol (THC)
strains include
Girl Scout Cookies (GSC), Kosher Kush, Ghost OG, Bruce Banner, Ghost Train
Haze,
Chemdawg, Ace of Spades, Afghani, Afgoo, AK-47, Alien OG, Alien Rock Candy,
Allen
Wrench, Animal Cookies, Sour Diesel, Skywalker, GG4, The White, Death Star,
White Fire
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OG, Kimbo Kush, Headband, Cherry Pie, Bubba Kush, SFV OG, LA Confidential and
Triangle
Kush. An exemplary high tetrahydrocannabivarin (THCV) strain includes Dutch
Treat.
[00099] Any part of the Cannabis plant may be used to produce cannabis
extracts. For
example, stems, leaves, seeds, flowers or a combination thereof can be used as
the starting
material for the extraction methods of the invention. In some aspects, one or
more parts of the
plant are used in producing cannabis extracts. Alternatively, all parts of the
plants may be used
in to produce cannabis extracts.
Cannabinoids
[000100] In some embodiments, the instant disclosure provides a cannabis
extract comprising
cannabinoids and methods of producing said cannabis extract.
[000101] Cannabinoids are a class of chemical compounds that act on the
cannabinoid
receptors, also known as the endocannabinoid system in cells. Cannabinoids
include
endocannabinoids, produced naturally in the body by animals;
phytocannabinoids, produced by
Cannabis and other plants; and synthetic cannabinoids, which are manufactured.
Phytocannabinoids, sometimes also referred to herein as cannabinoids, are a
structurally
diverse class of molecules that are derived from a common C21 precursor
(cannabigerolic acid,
or CBGA) or its C19 analog (cannabigerovaric acid, or CBGVA).
[000102] There are currently over 100 cannabinoids known to be produced by
Cannabis
plants, all of which can be included in the cannabis extract of the disclosure
and purified using
the methods described herein. Cannabinoids are described in, for example,
Brenneisen (2007).
Exemplary cannabinoids include Cannabichromenes such as Cannabichromene (CBC),
Cannabichromenic acid (CBCA), Cannabichromevarin (CBCV) and
Cannabichromevarinic
acid (CBCVA); Cannabicyclols such as Cannabicyclol (CBL), Cannabicyclolic acid
(CBLA)
and Cannabicyclovarin (CBLV); Cannabidiols such as Cannabidiol (CBD),
Cannabidiol
monomethylether (CBDM), Cannabidiolic acid (CBDA), Cannabidiorcol (CBD-C1),
Cannabidivarin (CBDV) and Cannabidivarinic acid (CBDVA); Cannabielsoins such
as
Cannabielsoic acid B (CBEA-B), Cannabielsoin (CBE) and Cannabielsoin acid A
(CBEA-A);
Cannabigerols such as Cannabigerol (CBG), Cannabigerol monomethylether (CBGM),
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Cannabigerolic acid (CBGA), Cannabigerolic acid monomethylether (CBGAM),
Cannabigerovarin (CBGV) and Cannabigerovarinic acid (CBGVA); Cannabinols and
cannabinodiols such as Cannabinodiol (CBND), Cannabinodivarin (CBVD),
Cannabinol
(CBN), Cannabinol methylether (CBNM), Cannabinol-C2 (CBN-C2), Cannabinol-C4
(CBN-
C4), Cannabinolic acid (CBNA), Cannabiorcool (CBN-C1) and Cannabivarin (CBV);
Cannabitriols such as 10-Ethoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9-
Dihydroxy-
delta-6a-tetrahydrocannabinol, Cannabitriol (CBT) and Cannabitriolvarin
(CBTV); Delta-8-
tetrahydrocannabinols such as Delta-8-tetrahydrocannabinol (A8-THC) and Delta-
8-
tetrahydrocannabinolic acid (A8-THCA); Delta-9-tetrahydrocannabinols such as
Delta-9-
tetrahydrocannabinol (THC), Delta-9-tetrahydrocannabinol-C4 (THC-C4), Delta-9-
tetrahydrocannabinolic acid A (THCA-A), Delta-9-tetrahydrocannabinolic acid B
(THCA-B),
Delta-9-tetrahydrocannabinolic acid-C4 (THCA-C4), Delta-9-
tetrahydrocannabiorcol (THC-
C1), Delta-9-tetrahydrocannabiorcolic acid (THCA-C1), Delta-9-
tetrahydrocannabivarin
(THCV) and Delta-9-tetrahydrocannabivarinic acid (THCVA); as well as 10-0xo-
delta-6a-
tetrahydrocannabinol (OTHC), Cannabichromanon (CBCF), Cannabifuran (CBF),
Cannabiglendol, Cannabiripsol (CBR), Cannbicitran (CBT), Dehydrocannabifuran
(DCBF),
Delta-9-cis-tetrahydrocannabinol (cis-THC), Tryhydroxy-delta-9-
tetrahydrocannabinol (tri0H-
THC) and 3,4,5,6-Tetrahydro-7-hydroxy-alpha-alpha-2-trimethy1-9-n-propy1-2,6-
methano-2H-
1-benzoxocin-5-methanol (OH-iso-HHCV).
[000103] The principle cannabinoid components present in plants of the
Cannabis species are
the cannabinoid acids, A9-tetrahydrocannabinolic acid (A9-THCA or THCA) and
cannabidiolic
acid (CBDA), with small amounts of the corresponding neutral cannabinoids,
respectively, i.e.,
A9-tetrahydrocannabinol (A9-THC or THC) and cannabidiol (CBD). Other
cannabinoid acids
include CBGA (cannabigerolic acid), CBCA (cannabichromenenic acid), CBGVA
(cannabigerovarinic acid), THCVA (tetrahydrocanabivarinic acid), CBDVA
(cannabidivarinic
acid), and CBCVA (cannabichromevarinic acid). Other neutral cannabinoids that
can be
included in the cannabis extracts described herein include CBN (cannabinol),
CBG
(cannabigerol), CBC (cannabichromene), CBL (cannabicyclol), CBV
(cannabivarin), THCV
(tetrahydrocannabivarin), CBDV (cannabidivarin), CBCV (cannabichromevarin),
CBGV
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(cannabigerovarin), CBGM (cannabigerol monomethylether), CBE (cannabielsoin),
and CBT
(cannabicitran).
[000104] Exemplary cannabinoids include metabolites of cannabinoids. For
example, the
metabolites of THC include 11-hydroxy-A9-tetrahydrocannabinol (11-0H-THC).
[000105] In some embodiments, the cannabis extract comprises at least 20%
cannabinoids, at
least 30% cannabinoids, at least 40% cannabinoids, at least 50% cannabinoids,
at least 60%
cannabinoids, at least 65% cannabinoids, at least 70% cannabinoids, at least
75% cannabinoids,
at least 80% cannabinoids, at least 85% cannabinoids, at least 90%
cannabinoids, at least 95%
cannabinoids, at least 96% cannabinoids, at least 97% cannabinoids, at least
98% cannabinoids
or at least 99% cannabinoids. In some embodiments, the cannabis extract
comprises at least
90% cannabinoids.
Terpenes
[000106] The instant disclosure provides cannabis extracts comprising
terpenes, and methods
of producing cannabis extracts comprising terpenes. In some embodiments, the
cannabis extract
comprises terpenes and cannabinoids.
[000107] Terpenes, sometimes referred to as terpenoids, are essential oil (EO)
components
present in numerous botanicals, including Cannabis, and form the largest group
of plant
chemicals, with 15-20,000 terpenes that have been fully characterized
(Langenheim, 1994).
Terpenes comprise a large group of compounds synthesized from Cio isoprene
subunits. The
European pharmacopoeia, Sixth Edition (2007), lists 28 E0s (Pauli and
Schilcher, 2010).
Terpenoids are pharmacologically versatile: they are lipophilic, interact with
cell membranes,
neuronal and muscle ion channels, neurotransmitter receptors, G-protein
coupled (odorant)
receptors, second messenger systems, and enzymes (Bowles, 2003; Buchbauer,
2010).
Monoterpenes (Cio) and sesquiterpenes (C15) are the classes most commonly
identified in
Cannabis spp. Terpenoids are the primary aromatic constituents of cannabis
resin, although
they constitute only a small percentage of organic solvent extracts (Elsohly
et al., 2007).
[000108] Without wishing to be bound by theory, it is thought that interplay
between the
effects of cannabinoids and other compounds derived from Cannabis such as
terpenes and/or
flavonoids, sometimes referred to as the "entourage effect" can enhance the
efficacy of
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cannabis extracts for the treatment of a variety of diseases and disorders.
For example, it is
thought that the terpene myrcene can enhance penetration across the blood
brain barrier, pinene
can counteract memory and cognition problems, while the combination of pinene,
myrcene,
and caryophyllene can help treat anxiety.
[000109] There are currently at least 80 to 100 terpenes that may be present
in Cannabis.
Exemplary terpenes produced by Cannabis that can be included in the cannabis
extracts
described herein comprise limonene, nerolidol, phytol, caryophyllene oxide,
linalool, a-pinene,
fl-pinene, eucalyptol, trans-nerolidol, humulene, delta-3-carene, camphene,
borneol, valencene,
geraniol, myrcene, terpinolene, 13-caryophyllene, selina-3 7(11)-diene,
guaiol, 10-epi-y-
eudesmol, fl-eudesmol, a-eudesmol, bulnesol, a-bisabolol, or a combination of
any of these. In
some embodiments, the terpenes in the cannabis extract comprise myrcene,
terpinolene,
caryophyllene, selina-3 7(11)-diene, guaiol, 10-epi-y-eudesmol, fl-eudesmol, a-
eudesmol,
bulnesol, a-bisabolol, a-humulene, a-pinene, limonene, linalool, or a
combination of any of
these.
[000110] Different Cannabis strains or varieties contain different terpene
compositions. For
example, strains such as Super Silver Haze, Skywalker and Rock Star produce of
beta-
caryophyllene. As a further example, strains such as Jack Herer, Strawberry
Cough, Blue
Dream, Island Sweet Skunk, Dutch Treat and Romulan produce pinenes. As a
further example,
strains such as Skunk XL, White Widow, and Special Kush produce myrcene. As
yet a further
example, strains such as Harle-Tsu, Pink Kush, Headband, OG Shark, and ACDC
produce a-
Bisabolol. The person of ordinary skill will be able to select a Cannabis
strain producing the
desired terpene(s) for use in making the extracts disclosed herein.
Flavonoids
[000111] In some embodiments, the instant disclosure provides cannabis
extracts comprising
flavonoids. In some embodiments, the cannabis extract comprises flavonoids and
cannabinoids.
In some embodiments, the cannabis extract comprises flavonoids, terpenes and
cannabinoids.
[000112] Flavonoids are secondary polyphenolic metabolites that commonly have
a ketone
group and yellowish pigments. In Cannabis, at least 20 flavonoids have been
identified, mainly
belonging to flavone and flavonol subclasses. Without wishing to be bound by
theory, it is
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thought that the flavonoids in Cannabis can exert a wide range of biological
effects, including
aiding in the efficacy of cannabis extracts for the treatment of diseases or
disorders through the
entourage effect.
[000113] Exemplary flavonoids that can be included in extracts of the
disclosure include, but
are not limited to, cannflavin A, cannflavin B, cannflavin C, vitexin,
isovitexin, apigenin,
kaempferol, quercetin, luteolin, orientin or a combination of any of these.
Methods of Making Cannabis Extracts
[000114] The cannabis extracts used in the compositions described herein can
be extracted
from cannabis plant material using any methods known in the art. Exemplary
methods include,
but are not limited to, lipid-based cold extraction, organic-solvent based
extraction,
supercritical fluid extraction, column chromatography, high performance liquid
chromatography (EIPLC) molecular distillation, or a combination thereof
[000115] Methods of purifying cannabis extracts will be readily apparent to
the person of
ordinary skill in the art.
[000116] Cannabis extracts can be made by exposing cannabis plants to carbon
dioxide,
butane, propane, alcohol, glycerin, and/or other solvents to leach compounds
from cannabis
plants.
[000117] Exemplary methods of making cannabis extracts are described in EP
1385595 B1
and US patent No. 7,344,736, the contents of each of which are incorporated by
reference in
their entireties. In some embodiments, cannabis extracts are made by
supercritical fluid
extraction. In supercritical fluid extraction, cannabis plant material or
crude extracts, for
example extracts precipitated using alcohol, are mixed with a suitable
solvent, and by
controlling temperature and pressure below the super-critical temperature and
pressure,
lipophilic or hydrophilic fractions rich in cannabinoids and other cannabis
components are
separated. Exemplary solvents used in supercritical fluid extraction include
carbon dioxide
(CO2).
[000118] In some embodiments, cannabis extracts are made using butane
extraction. In an
exemplary butane extraction protocol, cannabis plant material is saturated
with a solvent
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comprising butane and propane, and allowed to extract cannabinoids, terpenes
and other
molecules from the plant material. The plant material is then removed, and the
collected solvent
heated to remove the solvent via distillation and retain the cannabis extract.
Residual solvent
can be purged under vacuum.
[000119] In some embodiments, cannabis extracts are made using lipid-based
cold extraction
methods. Lipid-based cold extraction methods are described in W02020/028991,
the contents
of which are incorporated herein by reference. In an exemplary lipid-based
cold extraction
method, cannabis plant material is mixed with a cold lipid solvent in liquid
for a period of time,
for example between 10 and 60 minutes, at temperatures between 0 C to -40 C,
depending
upon the melting point of the lipid. The lipid solvent containing the cannabis
extract is then
separated from the cannabis plant material via centrifugation and/or
filtration. Suitable lipid
solvents include, but are not limited to, marine oil, fish oil, flax seed oil,
camelina oil, evening
primrose oil, black current oil, ahiflower seed oil, or a combination thereof
[000120] In some embodiments, cannabis extracts are made using organic solvent-
based cold
extraction methods. Organic solvent-based cold extraction methods are
described in
W02020/028992, the contents of which are incorporated herein by reference. In
an exemplary
organic-based cold extraction method, cannabis plant material is mixed with a
cold organic
solvent in liquid form for a period of time, for example between 10 and 60
minutes, at
temperatures between 0 C to -80 C, depending upon the organic solvent. The
organic solvent
containing the cannabis extract is then separated from the cannabis plant
material via
centrifugation and/or filtration. Suitable organic solvents include, but are
not limited to,
ethanol, methanol, acetone or ethyl acetate.
[000121] In some embodiments, cannabis extracts are made using a rosin press.
Rosin presses
use a combination of heat and pressure to extract rosin comprising
cannabinoids and other
bioactive molecules from cannabis plant material. Rosin presses are available,
for example
from Pure Pressure and other companies.
[000122] In some embodiments, cannabis extracts may be further purified by
chromatographic separation. High performance liquid chromatography (HPLC) is
an analytical
technique for determination and assay of constituents and can be used in
preparative mode to
produce quantities of concentrated fractions and individual components. EIPLC
uses pumps to
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pass a pressurized liquid solvent containing the cannabis extract through a
column filled with a
solid adsorbent material. Each component of the cannabis extract, such as
different terpenes,
flavonoids or cannabinoids, interacts slightly differently with the adsorbent
material, causing
different flow rates for the different components and leading to the
separation of the
components as they flow out of the column. However, HPLC is subject to
limitations of scale
as a production technique and there remains a need for additional methods of
separation to
produce large-scale quantities of cannabis extracts of sufficient quality for
formulation into
pharmaceutical dosage forms.
[000123] In some embodiments, distillation and/or sublimation can be used to
purify cannabis
extracts of the instant disclosure. Distillation and sublimation have been
used to separate
components of plant medicines which have boiling points at or around the
temperature at which
water boils at atmospheric pressure (100 C). Separation by distillation is a
physical process
widely used in the preparation of essential oils. For example, GB 635,121
describes a process
for the preparation of extracts from aromatic plants by distillation with the
help of a hot gas,
preferably under high vacuum. As a further example, WO 99/11311 describes a
vaporizer for
inhalation and a method for the extraction of active ingredients from a crude
natural product.
This method utilizes an ascending stream of hot air, or a heated inert gas
stream, to volatilize
components from the natural product. The resultant vapor may then be inhaled
by a user. As yet
a further example, W000/25127 is concerned with a method of preparing
tetrahydrocannabinol
using extraction of plant material with a non-polar solvent followed by vacuum
distillation and
collection of a constant boiling fraction. Additional distillation steps and
chromatographic
steps, including HPLC, reverse phase HPLC and flash chromatography, may be
performed.
[000124] In some embodiments, molecular distillation can be used to purify
cannabis extracts
of the instant disclosure. Molecular distillation, sometimes called short path
distillation, is a
separation technique that separates compounds through a process of slow
thermal heating. The
compounds in cannabis extracts, such as cannabinoids, terpenes and flavonoids,
have different
vapor pressure points (boiling points). Through precise temperature control of
the distillation
process, molecular distillation can separate a cannabis extract into one or
more high-purity
fractions. In exemplary embodiments, the final materials produced through
short path
distillation include one or more cannabinoids, one or more terpenes, and
optionally, any
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leftover waxes, sugars, and heavy residues. In some embodiments, the molecular
distillation
comprises more than one round of molecular distillation.
[000125] In some embodiments, cannabis extracts can be purified using column
chromatography. Column chromatography is a method use to separate compounds
based on
differential absorption of the compounds to the adsorbent packed in a column.
The compounds,
such as different terpenes, flavonoids and cannabinoids move through the
column at different
rates, allowing them to be separated into fractions. The column chromatography
can be carried
out using any known packing material including, for example, silica or alumina
for normal
phase operation or Cil3 or CI3 bonded phase silica for reversed phase
operation. Elution of the
normal phase chromatography column is carried out with solvents having an
increasing
polarity. Non-polar solvents include the lower straight chain and branched
chain alkanes,
including, for example, pentane, hexane, isooctane and petroleum ether. More
polar solvents
include various organic ethers, alcohols, esters or ketones, including, for
example dialkyl
ethers, lower alkyl acetates, lower dialkyl ketones and lower alkanols.
Illustrative polar
solvents include, for example, acetone, ethylacetate, diethylether and
isopropyl alcohol. The
ratio of non-polar solvent to polar solvent can vary between 100:0 to 80:20.
[000126] Methods of formulating cannabis extracts in suitable solvents for
combining with
the lipid-based carriers disclosed herein will be readily apparent to the
person of ordinary skill
in the art. For example, an undesired solvent such as ethanol or butane can be
removed by
evaporation, and the resulting cannabis extract precipitate re-dissolved in a
suitable solvent,
such as marine oil, fish oil, flax seed oil, camelina oil, evening primrose
oil, black current oil,
ahiflower seed oil, or a combination thereof
Bleaching
[000127] In some embodiments, the methods described herein comprise bleaching
the
cannabis extract. As used herein, "bleaching" refers to a process of removing
undesired minor
impurities from a botanical extract, such as color pigments, free fatty acids,
peroxides,
undesired odor causing compounds and non-fatty materials.
[000128] In some embodiments, bleaching comprises contacting the botanical
extract with a
bleaching agent. Exemplary bleaching agents include natural earth clay,
bentonite, acid
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activated clay, silica gel, diatomaceous earth, bleaching earth, activated
carbon, mixtures of
magnesium oxide and alumina zeolitic, or combinations thereof For example, the
botanical
extract can be filtered through a cake of bleaching agent and a filter using a
vacuum.
Winterizing and De-waxing
[000129] In some embodiments, the methods of preparing a cannabis extract
comprise
winterization and/or de-waxing. Winterization and de-waxing are methods to
remove undesired
cannabis lipids and waxes from cannabis extracts. Winterization can be
achieved by dissolving
a non-polar substance (e.g., the cannabinoid extract) into a polar solvent
(e.g. ethanol) at sub-
zero temperatures. This separates waxes and lipids from the cannabinoid
extract, forcing them
to collect at the top of the mixture for easy filtration.
[000130] An exemplary winterization method is described in US 7,344,736.
Ethanol is added
to the cannabis extract in the ratio of 2:1 ethanol volume to weight. The
ethanolic solution is
then cooled to ¨20 C 5 C and held at this temperature for approximately 48
hours. On
completion of the winterization, the precipitated waxes and lipids are removed
by cold filtration
through a 20 pm filter.
[000131] De-waxing also uses low temperatures to separate waxes and lipids
from cannabis
extract. In de-waxing, cannabis extract mixed with a solvent such as butane is
cooled to low
temperatures (e.g. -20 C or below) which makes the waxes and lipids insoluble
in the butane
solution. Once the waxes and undesired lipids have separated from the solvent,
the mixture is
passed through a variety of micron screens, effectively filtering out all
undesired waxes and
lipids. An exemplary de-waxing protocol comprises chilling the cannabis
extract and butane
composition to low temperatures, then running the composition through a
Buchner funnel that
is attached to a passive vacuum, thus filtering out waxes and lips and
producing a purer final
product. The filtered product is then passed to a heated chamber where the
butane can be
removed through evaporation.
Decarboxylation
[000132] In some embodiments, cannabis plant material used in the extraction
methods
described herein is decarboxylated. Decarboxylation is a chemical reaction
that converts an
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acid to a phenol, and releases carbon dioxide (CO2), thereby removing a carbon
atom from a
carbon chain. Most cannabinoids exist as acids and neutral (i.e.
decarboxylated) forms.
Phytocannabinoids are synthesized in the plant as acid forms. Some
decarboxylation does occur
in the cannabis plant. However, decarboxylation increases significantly after
the plant is
harvested, and the kinetics of decarboxylation increase at higher temperatures
than found in
vivo.
[000133] All methods of decarboxylation known in the art are envisaged as
within the scope
of the instant disclosure. Exemplary decarboxylation methods are described in
US 7,344,736,
the contents of which are incorporated by reference in their entirety.
[000134] The decarboxylation step may be carried out prior to or after
extraction of the
cannabis plant material.
[000135] In some embodiments, the decarboxylation step is carried out prior to
extraction and
is conducted by heating the cannabis plant material to temperatures and for
times which ensure
at least 95% conversion of the acid cannabinoids from the acid form to their
neutral form, while
ensuring thermal degradation of THC to CBN is less than 10%.
[000136] Decarboxylation of cannabinoid acids is a function of time and
temperature, thus at
higher temperatures a shorter period of time will be taken for complete
decarboxylation of a
given amount of cannabinoid acid. In selecting appropriate conditions for
decarboxylation
consideration must, however, be given to minimizing thermal degradation of the
desirable,
pharmacological cannabinoids into undesirable degradation products, for
example thermal
degradation of THC to cannabinol (CBN).
[000137] In some embodiments, decarboxylation is carried out in a multi-step
heating process
in which the plant material is first heated to a first temperature for a first
(relatively short) time
period to evaporate off retained water and allow for uniform heating of the
plant material; and
second the temperature is increased to a second temperature for a second time
period (typically
longer than the first time period) until at least 95% conversion of the acid
cannabinoids to their
neutral form has occurred.
[000138] In some embodiments, the first step is conducted at a temperature in
the range of
100 C to 110 C for 10 to 20 minutes. In some embodiments, the first
temperature is about
105 C and the first time period is about 15 minutes.
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[000139] If the plant material is derived from cannabis plants having a high
CBD content, the
second temperature can be in the range from 115 C to 125 C, for example about
120 C and the
second time period is in the range from 45 to 75 minutes, for example about 60
minutes. In
some embodiments, the second temperature is in the range from 135 C to 145 C,
for example
140 C and the second time period is in the range from 15 to 45 minutes, for
example about 30
minutes.
[000140] If the plant material is derived from cannabis plants having a high
THC content, the
second temperature is can be in the range of 115 C to 125 C, for example 120
C, and the
second time period can be in the range of 45 minutes to 75 minutes, for
example about 60
minutes. In some embodiments, the second temperature is in the range of 100 C
to 110 C, for
example 105 C, and the second time period is in the range of 60 to 120
minutes.
[000141] In some embodiments, the decarboxylation step is conducted at
temperatures and for
times which ensure at least 97% conversion of the acid cannabinoids to their
neutral form,
while ensuring thermal degradation of THC to CBN is less than 5%.
[000142] In some embodiments, decarboxylation is carried out in 2 steps, for
example 105 C
for 15 minutes, and then at 110 C for about 40 to 75 minutes.
[000143] In some embodiments, decarboxylation is carried out in a single step
heating
process in which the plant material is heated to between about 115 C to 145 C.
In some
embodiments, decarboxylation is carried out in a single step heating process
in which the plant
material is heated to between about 110 C to 145 C. In some embodiments,
decarboxylation is
carried out at about 110 C or 115 C. In some embodiments the plant material is
heated to
between about 110 C to 145 C for less than 15 minutes, less than 30 minutes,
less than 45
minutes, less than 60 minutes, less than 75 minutes, less than 90 minutes,
less than 105 minutes
or less than 120 minutes. In some embodiments the plant material is heated to
between about
110 C to 145 C for less than one hour. In some embodiments the plant material
is heated to
between about 110 C to 145 C for between about 30 and 60 minutes.
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Antioxidants
[000144] The disclosure provides compositions comprising a cannabis extract, a
lipid-based
carrier and an antioxidant.
[000145] In some embodiments, the antioxidant is a fat-soluble antioxidant.
Antioxidants are
compounds that inhibit oxidation, a chemical reaction that can produce free
radicals, which can
cause cellular damage.
[000146] In some embodiments, the antioxidant comprises alpha tocopherol, a
mixture of
tocopherols, or rosemary extract. Exemplary tocopherols include d-a-tocopheryl
acetate, d-a-
tocopheryl acid succinate, d-13-tocopherol, d-13-tocopherol, d-y-tocopherol, d-
6-tocopherol, d-a-
tocotrienol, d-I3-tocotrienol, d-y-tocotrienol, d-6-tocotrienol, dl-a-
tocopherol, dl-a-tocopheryl
acetate, dl-a-tocopheryl calcium succinate, dl-a-tocopheryl nicotinate, dl-a-
tocopheryl
linoleate/oleate and all other possible stereo isomeric forms of the above
compounds, and are
sometimes referred to as "Vitamin E." Additional antioxidants include
astaxanthin, beta-
carotene, carotenoids, and Vitamin A.
Methods of making Cannabis Extract Compositions
[000147] The disclosure provides methods of making the compositions described
herein,
comprising (a) providing a cannabis extract; and (b) mixing the cannabis
extract with a lipid-
based carrier. In some embodiments, the lipid based carrier comprises omega-3
fatty acids,
monoacylglycerides, diacylglycerides, triglycerides, phospholipids or a
combination thereof
[000148] In some embodiments, the methods comprise mixing the cannabis extract
and the
lipid carrier with one or more antioxidants.
[000149] In some embodiments, cannabis extract comprises a liquid or a resin.
In some
embodiments, cannabis extract comprises a liquid. In some embodiments,
cannabis extract
comprises a resin. In some embodiments, cannabis extract comprises a powder.
[000150] In some embodiments, either the cannabis extract, the lipid-based
carrier or both, is
formulated or diluted with a pharmaceutically acceptable carrier, diluent or
excipient. The
pharmaceutically acceptable carrier, diluent or excipient can be a liquid, for
example a liquid
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PCT/CA2020/051081
comprising fish oil, flax seed oil, camelina oil, evening primrose oil, black
current oil,
ahiflower seed oil, or a combination thereof
[000151] In some embodiments, the lipid-based carrier comprises fish oil,
krill oil, flax seed
oil, or a derivative thereof In some embodiments, fish oil, the krill oil or
the flax seed oil has
been processed to increase the percentage of MAG, DAG, triglycerides,
phospholipids or a
combination thereof in the fish oil, the krill oil or the flax seed oil as
described herein.
[000152] In some embodiments, the cannabis extract is mixed with the lipid-
based carrier at a
ratio of about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8,
about 1:9, about 1:9.5,
about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about
1:16, about 1:17,
about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about
1:24 or about 1:25
cannabis extract to lipid-based carrier. In some embodiments, the cannabis
extract is mixed
with the lipid-based carrier at a ratio of about 1:7, about 1:8, about 1:9,
about 1:9.5, about 1:10,
about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about
1:17, about 1:18,
about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24 or
about 1:25 cannabis
extract to lipid-based carrier.
Formulations
[000153] The disclosure provides compositions comprising (a) a cannabis
extract comprising
at least one cannabinoid, and (b) a lipid-based carrier.
[000154] In some embodiments, the lipid-based carrier comprises omega-3 fatty
acids,
monoacylglycerides, diacylglycerides and phospholipids. In some embodiments,
the omega-3
fatty acids comprise omega-3 monoacylglycerides, omega-3 diacylglycerides,
omega-3
phospholipids or a combination thereof
[000155] In some embodiments, the composition comprises at least one
monoacylglyceride
(MAG).
[000156] In
some embodiments, at least 4%, at least 5%, at least 6%, at least 10%, at
least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45% or at least
50% of the total glycerides in the composition comprise monoacylglycerides
(MAG).
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[000157] In some embodiments, at least 4% of the total glycerides in the
composition
comprise MAG. In some embodiments, at least 30% of the total glycerides in the
composition
comprise MAG.
[000158] In some embodiments, between about 4% to 50%, about 10% to 50%, about
20% to
50%, about 25% to 50%, about 30% to 50%, about 35% to 50%, about 40% to 50%,
about 45%
to 50%, about 4% to 40%, about 10% to 40%, about 20% to 40%, about 30% to 40%,
about 4%
to 35%, about 10% to 35%, about 20% to 35%, about 30% to 35%, about 4% to 30%,
about
10% to 30% or about 20% to 30%, of the total glycerides in the composition
comprise MAG.
[000159] In some embodiments, the composition comprises at least one
diacylglyceride
(DAG).
[000160] In some embodiments, at least 1%, at least 3%, at least 5%, at least
7% at least 10%,
at least 20%, at least 30%, at least 40%, at least 45%, at least 47%, at least
50%, at least 60%,
at least 70%, at least 80% or at least 85% of the glycerides in the
composition are
diacylglycerides (DAG). In some embodiments, at least 1%, at least 3%, at
least 5%, at least
7%, at least 10%, at least 20%, at least 30%, at least 40%, at least 45%, at
least 47%, at least
50%, at least 60%, or at least 70% of the glycerides in the composition are
diacylglycerides
(DAG). In some embodiments, at least 40%, at least 45%, at least 47%, at least
50%, at least
60%, at least 70%, at least 80% or at least 85% of the glycerides in the
composition comprise
DAG. In some embodiments, at least 47% of the glycerides in the composition
comprise DAG.
[000161] In some embodiments, the ratio of MAG:DAG in the composition is about
0.5:1,
about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 1.1:1,
about 1.2:1, about
1.3:1, about 1.4:1, about 1.5:1, about 1.61, about 1.7 :1, about 1.8:1, about
1.9:1, about 2:1,
about 2.1:1, about 2.2:1, about 2.3:1, about 2.4:1, about 2.5:1 or about 3:1.
[000162] In some embodiments, the composition comprises phospholipids. In some
embodiments, the composition comprises phosphatidylcholine,
phosphatidylinositol, and
phosphatidylethanolamine phospholipids and phosphatidylcholine is at least 75%
of the total
phospholipid content. In some embodiments, the phospholipids are at least 20%,
at least 25%,
at least 35%, at least 40%, at least 50%, at least 60% or at least 70% of the
lipids in the
composition. In some embodiments, the composition comprises phospholipids and
triglycerides. In some embodiments, the phospholipids and triglycerides are
present at a ratio of
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about 1:1. In some embodiments, the ratio of triglycerides to phospholipids is
about 1:1.3. In
some embodiments, the ratio of triglycerides to phospholipids is about 1:1.7.
In some
embodiments, the ratio of triglycerides to phospholipids is about 1:3. In some
embodiments, the
ratio of triglycerides to phospholipids is about 1:4. In some embodiments, the
ratio of
triglycerides to phospholipids is about 1:7.
[000163] In some embodiments, the at least one cannabinoid comprises
A9tetrahydrocannabinol (THC), cannabidiol (CBD), tetrahydrocannabinolic acid
(THCA),
cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabichromenenic acid
(CBCA),
cannabigerovarinic acid (CBGVA), tetrahydrocanabivarinic acid (THCVA),
cannabidivarinic
acid (CBDVA), cannabichromevarinic acid (CBCVA), cannabinol (CBN),
cannabigerol
(CBG), cannabichromene (CBC), cannabicyclol (CBL), cannabivarin (CBV),
tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabichromevarin
(CBCV),
cannabigerovarin (CBGV), cannabigerol monomethylether (CBGM), cannabielsoin
(CBE),
cannabicitran (CBT), or a combination thereof
[000164] In some embodiments, the at least one cannabinoid comprises a
combination of
THC and CBD.
[000165] In some embodiments, the at least one cannabinoid comprises a
combination of
THC, THCA, CBD and CBDA.
[000166] In some embodiments, the composition comprises about 2% to about 50%
cannabinoids, about 2% to about 45% cannabinoids, about 2% to about 40%
cannabinoids,
about 2% to about 30% cannabinoids, about 2% to about 20% cannabinoids, about
2% to about
15% cannabinoids, 5% to about 50% cannabinoids, about 5% to about 45%
cannabinoids,
about 5% to about 40% cannabinoids, about 5% to about 30% cannabinoids, about
5% to about
20% cannabinoids, about 5% to about 15% cannabinoids, about 10% to about 50%
cannabinoids, about 10% to about 45% cannabinoids, about 10% to about 40%
cannabinoids,
about 10% to about 30% cannabinoids, about 10% to about 20% cannabinoids or
about 10% to
about 15% cannabinoids.
[000167] In some embodiments, the composition comprises about 2% to 20%
cannabinoids.
[000168] In some embodiments, the composition comprises about 5% to 20%
cannabinoids.
[000169] In some embodiments, the composition comprises about 2% to 50%
cannabinoids.
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[000170] In some embodiments, the composition comprises at least one
cannabinoid, at least
one terpene, and a lipid carrier. In some embodiments, the composition
comprises at least one
cannabinoid, at least one terpene, at least one flavonoid and a lipid carrier.
In some
embodiments, the terpene comprises myrcene, terpinolene, 13-caryophyllene,
selina-3 7(11)-
diene, guaiol, 10-epi-y-eudesmol, 13-eudesmol, a-eudesmol, bulnesol, a-
bisabolol, a-humulene,
a-pinene, limonene, linalool, or a combination thereof In some embodiments,
the flavonoid
comprises cannflavin A, cannflavin B, cannflavin C, vitexin, isovitexin,
apigenin, kaempferol,
quercetin, luteolin, orientin or a combination thereof
[000171] In some embodiments, the composition comprises an antioxidant such as
alphatocopherol, a mixture of tocopherols, or rosemary extract.
[000172] In some embodiments, the composition comprises a pharmaceutically
acceptable
carrier, diluent or excipient. The pharmaceutically acceptable carrier,
diluent or excipient can
be a liquid, for example a liquid comprising fish oil, flax seed oil, camelina
oil, evening
primrose oil, black current oil, ahiflower seed oil, or a combination thereof
Properties of
pharmaceutically acceptable carriers are described in Table 1 below:
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TABLE 1: Fatty acid profiles of exemplary oils with a freezing point below -5
C
Oil Type Solvent Name to-3 MAG; PUFA/SFA Freezing
(as % FA) DAG2 Index Point
Animal Oil EE Fish Oil 88 1-3% MAG; 46.3 -40 C
RTG Fish Oil 75 1-40% DAG>100
Flax seed oil Omega Nutrition flax seed 55 0.2-3%
MAG; 8.9 -24 C
(from different oil 0.5-7% DAG
sources) TAFOODs flax seed oil 57 10.7
Shape Foods High ALA 66 10.1
Flax oil
Shape Foods Organic Cold 57 9.1
press
Camelina oil1 Camelina oil 35 7.3 -15 C
EPO EPO >9 10.3 -20 C
Ahiflower Natures Crops Ahiflower oil 66 11.5 -20 C
seed oil
Hemp seed oil Hemp seed oil Chii 18 8.2 -8 C
Black currant Black currant oil 15 9.1 -20 C
oil
Abbreviations: co, omega; ALA, alpha-linolenic acid; DAG, diacylglyceride;
EPO, evening
primrose oil; FA, fatty acid; MAG, monoacylglyceride; PUFA, polyunsaturated
fatty acid;
RTG, re-esterified triglyceride; SFA, saturated fatty acid
1 Data from Health Canada; 2indicates percent glycerides that are MAG and that
are DAG
[000173] In some embodiments, the composition is formulated for oral
administration.
An oral composition according to the instant disclosure may be in any of the
dosage forms
which are generally used for dietary supplements, such as liquids, gels,
powders, tablets,
caplets, capsules, gelcaps, food additives, drops, beverages, pills, lozenges,
rinses, pastes, gums
and soft gels.
[000174] Any pharmaceutically acceptable carrier, diluent or excipient known
in the art can
be used in the cannabis extract compositions described herein. Examples of
pharmaceutically
acceptable carriers, diluents and excipients for oral delivery include: sodium
bicarbonate
solutions and similar diluents which neutralize stomach acid or have similar
buffering capacity,
glycols, oils or emulsions; and include formulations in the form of gels,
pastes and viscous
colloidal dispersions. The cannabis extract compositions may be presented in
capsule, tablet,
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slow release or elixir form or as a gel or paste. Furthermore, the cannabis
extract compositions
may be presented as a food or drink.
[000175] Suitable carriers or diluents illustratively include, but are not
limited to, either
individually or in combination, lactose, including anhydrous lactose and
lactose monohydrate;
starches, including directly compressible starch and hydrolyzed starches;
mannitol; sorbitol;
xylitol; dextrose and dextrose monohydrate; dibasic calcium phosphate
dihydrate; sucrose-
based diluents; confectioner's sugar; monobasic calcium sulfate monohydrate;
calcium sulfate
dihydrate; granular calcium lactate trihydrate; dextrates; inositol;
hydrolyzed cereal solids;
amylose; celluloses including microcrystalline cellulose, food grade sources
of alpha- and
amorphous cellulose, powdered cellulose, and hydroxypropylmethylcellulose
(HPMC);
calcium carbonate; glycine; bentonite; block co-polymers;
polyvinylpyrrolidone; and the like.
[000176] Cannabis extract compositions of the disclosure optionally comprise
one or more
pharmaceutically acceptable disintegrants as excipients, particularly for
tablet formulations.
Suitable disintegrants include, but are not limited to, either individually or
in combination,
starches, including sodium starch glycolate and pregelatinized corn starches,
celluloses such as
purified cellulose, microcrystalline cellulose, methylcellulose,
carboxymethylcellulose and
sodium carboxymethylcellulose, croscarmellose sodium, alginates, crospovidone,
and gums
such as agar, guar, locust bean, karaya, pectin and tragacanth gums.
[000177] Cannabis extract compositions of the disclosure optionally comprise
one or more
pharmaceutically acceptable binding agents or adhesives as excipients,
particularly for tablet
formulations. Such binding agents and adhesives preferably impart sufficient
cohesion to the
powder being tableted to allow for normal processing operations such as
sizing, lubrication,
compression and packaging, but still allow the tablet to disintegrate and the
composition to be
absorbed upon ingestion. Suitable binding agents and adhesives include, but
are not limited to,
either individually or in combination, acacia; tragacanth; sucrose; gelatin;
glucose; starches
such as, but not limited to, pregelatinized starches; celluloses such as, but
not limited to,
methylcellulose and carmellose sodium Tylose; alginic acid and salts of
alginic acid;
magnesium aluminum silicate; polyethylene glycol (PEG); guar gum;
polysaccharide acids;
bentonites; povidone, for example povidone K-15, K-30 and K-29/32;
polymethacrylates;
hydroxypropylcellulose; and ethylcellulose.
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[000178] Polymeric binding agents can have varying molecular weight, degrees
of
crosslinking, and grades of polymer. Polymeric binding agents can also be
copolymers, such as
block copolymers that contain mixtures of ethylene oxide and propylene oxide
units. Variation
in these units' ratios in a given polymer affects properties and performance.
Examples of block
co-polymers with varying compositions of block units are Poloxamer 188 and
Poloxamer 237
(BASF Corporation).
[000179] Cannabis extract compositions of the disclosure optionally comprise
one or more
pharmaceutically acceptable wetting agents as excipients. Non-limiting
examples of surfactants
that can be used as wetting agents in cannabis extract compositions of the
disclosure include
quaternary ammonium compounds, for example benzalkonium chloride, benzethonium
chloride
and cetylpyridinium chloride, dioctyl sodium sulfosuccinate, polyoxyethylene
alkylphenyl
ethers, for example nonoxynol 9, nonoxynol and octoxynol 9, poloxamers
(polyoxyethylene
and polyoxypropylene block copolymers, polyoxyethylene fatty acid glycerides
and oils, for
example polyoxyethylene caprylic/capric mono- and diglycerides,
polyoxyethylene, castor oil
and polyoxyethylene, hydrogenated castor oil; polyoxyethylene alkyl ethers,
for example
polyoxyethylene cetostearyl ether, polyoxyethylene fatty acid esters, for
example
polyoxyethylene stearate, polyoxyethylene sorbitan esters, for example
polysorbate and
polysorbate, Tween 80, propylene glycol fatty acid esters, for example
propylene glycol
laurate, sodium lauryl sulfate, fatty acids and salts thereof, for example
oleic acid, sodium
oleate and triethanolamine oleate, glyceryl fatty acid esters, for example
glyceryl monostearate,
sorbitan esters, for example sorbitan monolaurate, sorbitan monooleate,
sorbitan monopalmitate
and sorbitan monostearate, tyloxapol, and mixtures thereof
[000180] Cannabis extract compositions of the disclosure optionally comprise
one or more
pharmaceutically acceptable lubricants (including anti-adherents and/or
glidants) as excipients.
Suitable lubricants include, but are not limited to, either individually or in
combination,
glyceryl behapate (Compritol 888); stearic acid and salts thereof, including
magnesium,
calcium and sodium stearates; hydrogenated vegetable oils; colloidal silica;
talc; waxes; boric
acid; sodium benzoate; sodium acetate; sodium fumarate; sodium chloride; DL-
leucine; PEG
Carbowax; sodium oleate; sodium lauryl sulfate; and magnesium lauryl sulfate.
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[000181] Suitable anti-adherents include, but are not limited to, talc,
cornstarch, DL-leucine,
sodium lauryl sulfate and metallic stearates.
[000182] Glidants can be used to promote powder flow of a solid formulation.
Suitable
glidants include, but are not limited to, colloidal silicon dioxide, starch,
talc, tribasic calcium
phosphate, powdered cellulose and magnesium trisilicate. Colloidal silicon
dioxide is
particularly preferred. Other excipients such as colorants, flavors and
sweeteners are known in
the pharmaceutical art and can be used in Cannabis extract compositions of the
instant
disclosure. Tablets can be coated, for example with an enteric coating, or
uncoated.
Compositions of the invention can further comprise, for example, buffering
agents.
[000183] Compositions of the instant disclosure may also contain additives,
such as water,
alcohols, oils (mineral, vegetable, animal and synthetics), glycols,
colorants, preservatives,
emulsifiers, gelling agents, gums, esters, hormones, steroids, antioxidants,
silicones, polymers,
fragrances, flavors, other active ingredients, acids, bases, buffers,
vitamins, minerals, salts,
polyols, proteins and their derivatives, essential oils, other enzymes, co-
enzymes and extracts,
surfactants, detergents, soaps, anionics, non-ionics, ionics, waxes, lipids,
stabilizers, fillers,
celluloses, glycans, amines, solubilizers, thickeners, sugars and sugar
derivatives, ceramides,
sweeteners and the like, so long as such additives do not defeat the
objectives of the present
invention.
[000184] Cannabis extract compositions of the disclosure may be formulated for
transmucosal
administration. For example, transmucosal administration can encompass oral
formulations for
buccal administration, and aerosol sprays for nasal administration and/or
inhalation.
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terpene content
during the growth of Cannabis sativa plants from different chemotypes. Journal
of
Natural Products, 79(2): 324-331.
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3. Huestis, M.A. (2007). Human cannabinoid pharmacokinetics. Chemsitry &
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4. Zgair, A., et al. (2016). Dietary fats and pharmaceutical lipid excipients
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14. Buchbauer G. (2010). Biological activities of essential oils. In: Baser,
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EXAMPLES
Example 1: Bioavailability of cannabis extracts formulated with
monoacylglyceride-rich
omega-3 fatty acid oil derived from fish
[000185] Cannabis extracts formulated in different lipid carriers were
administered to rats via
oral gavage, and the plasma concentrations of exemplary cannabinoids THC and
CBD were
measured.
[000186] The animal study was performed in accordance with the guidelines for
the Care and
Use of Laboratory Animals and approved by an Animal Care Committee. Male
Sprague-
Dawley rats (250-300 g bw) of 8 to 10 weeks of age were used for this study.
Animals were
maintained under controlled environmental conditions and a 12-hour light/dark
cycle. The
study was conducted after rats were allowed 4 days of acclimatization with
free access to water
and standard rat chow. A jugular vein catheter (0.02 in I.D. x 0.037 in 0.D.)
was surgically
inserted 24 hours prior to dosing to minimize possible anesthetic effects.
This catheter was used
for intravenous injections (IV group, n=4) and to withdraw blood (all groups).
Animals
(n=5/group) were randomly assigned to one of the oral (gavage) groups. The
dose groups were
as follows:
= 1:1 THC:CBD IV in lipid-free solution (propyleneglycol-ethanol-sterile
water
(80:10:10 v/v/v); 4 milligrams per kilogram of body weight, or mg/kg bw)
= 1:1 THC:CBD oral in medium chain triglyceride (MCT) coconut oil (12 mg/kg
bw)
= 1:1 THC:CBD oral in flax seed oil (TG-03) (12 mg/kg bw)
= 1:1 THC:CBD oral in MAG Fish Oil (MAG-03) (12 mg/kg bw)
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[000187] Injection and gavage volumes were determined according to the weight
of the
animals (1 milliliter per kilogram body weight (mL/kg bw)). Animals were
placed in a
containment chamber prior to IV injection or immediately after gavage to
facilitate blood
sampling. Blood samples (0.2 mL to obtain about 0.1 mL of plasma after
centrifugation) were
taken at 15, 30, 60 minutes and 2, 4, 6, 8 and 24 hours following oral
administration and at 0, 5,
15, 30, 60 minutes and 2, 4, 6, 8 hours following IV administration. Blood
losses were
compensated by injecting 0.9% saline solution (1 mL) after the 2-h time point
via the sampling
catheter. Blood sample collection was made in K2-EDTA microtainer tubes and
immediately
placed on crushed ice before being centrifuged at 8,000 rpm for 10 min at 4 C
to isolate plasma
from blood cells. The resultant plasma was then separated and transferred to
polypropylene
tubes, and immediately frozen at -80 C. The final blood sample was taken at 8h
(IV) or 24h
(oral) immediately after abdominal aorta sectioning (under anesthesia).
[000188] Analyses of cannabinoids, including CBD and THC, were performed on a
Sciex
Qtrap 6500+ equipped with a microflow liquid chromatography. An UPLC HSS-T3
column
(100 mm * lmm, 1.8 [tm, equipped with a 0.2 [tm fitted pre-filter) was used
for the
chromatographic separation. The solvent flow rate was set to 50 IlL/min and
the column
temperature kept at 40 C. The mobile phases were 0.1% formic acid in water and
0.1% formic
acid in methanol. The concentrations of CBD and THC in rat plasma were
determined
according to previous validated method using liquid-liquid extraction.
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TABLE 2: Pharmacokinetic parameters of CBD and THC after intravenous (IV)
bolus and
oral (gavage) administration of cannabis extract in various carrier oils
Formulation Clearance Vd AUCim Tmax Cmax fabs frel Flax
frel MCT
mL/h/lxg mL/kg 1)*(ng/mL) h ng/mL
Intravenous administration
CBD IV 6991 26616 340 18
THC IV 10452 32764 231 18
Oral administration
CBD MCT 428 45 4.0 1.0 114 24 42 4 142
21 100
CBD TG-03 310 19$ 4.4 1.2 78 12 30 2$
100 78 13
CBD MAG-03 - 786 122* 4.0 1.0 215 48 77 12* 254
391 206 57
THC MCT 1243 64 4.0 0.6 235 10 180 9
115 8 100
THC TG-03 1090 46 3.6 0.7 222 21 158 7
100 89 6
THC MAG-03 - 2382 532 4.4 0.4 507 159
344 77 213 36 197 48
[000189] Table 2 shows the results from administering an IV bolus of
cannabinoids in lipid-
free solution (4 mg/kg, n=4) and oral administration of cannabinoid
formulations (12 mg/kg,
n=5/group) via different carrier oils (monoacylglyceride-rich fish oil, MAG-
03; flax seed oil,
TG-03; medium chain triglycerides from coconut oil, MCT) in rats. Data was
taken from 0 to 8
hours post-dosage for IV group and 1 to 8 hours for oral group. Values are
expressed as mean
the Standard Error of the Mean (SEM). Statistical analysis was performed using
unpaired two-
tailed Student's t-test and one-way analysis of variance (ANOVA), where
appropriate. *
signifies p < 0.05 when compared to TG-03 and MCT oils; t signifies p < 0.05
when compared
to TG-03 oil; $ signifies p < 0.05 when compared to MCT oil. Abbreviations:
AUCrer, total
area under the curve; CBD, cannabidiol; C., maximum serum concentration; f
-abs, absolute
bioavailability; frei, relative bioavailability; THC, tetrahydrocannabinol;
T., time to reach
maximum plasma concentration; Vd, volume of distribution.
[000190] FIG. 1 shows the plasma concentration-time profile of cannabidiol
(CBD) from
cannabis extract administered to rats using the methods described above. In
FIG. 1, oral
formulations (12 mg/kg, n=5/group) of cannabis extract in different carrier
oils were
administered to rats via oral gavage and the plasma concentration of CBD from
0 to 24 hours
post-administration was measured. Values are expressed as mean SEM. The peak
plasma
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concentration of CBD following intravenous administration of cannabis extract
in lipid-free
solution was measured as 1193 290 ng/mL at 5 minutes (data not shown).
[000191] FIGS. 2A-2D summarize the pharmacokinetic parameters of CBD
absorption. Oral
formulations (12 mg/kg, n=5 rats/group) of cannabis extract in different
carrier oils was
administered to rats as above, and the CBD plasma concentration was measured
at 1 to 8 hours
post-administration, as described. FIG. 2A shows the area under the curve
(AUC) generated by
plotting CBD plasma concentration (in ng/mL) versus time in units of hours
(h). FIG. 2B shows
the maximum CBD plasma concentration. FIG. 2C shows absolute CBD
bioavailability; and
FIG. 2D shows the relative CBD bioavailability. Values are expressed as mean
SEM.
Statistical analysis was performed using unpaired two-tailed Student's t-test
and one-way
ANOVA, where appropriate. * signifies p < 0.05 when compared to TG-03 (flax
seed) and
medium chain triglyceride (MCT) oils; t signifies p < 0.05 when compared to TG-
03 (flax
seed) oil; $ signifies p < 0.05 when compared to MCT oil.
[000192] FIG. 3 shows the plasma concentration-time profile of
tetrahydrocannabinol (THC)
administered to rats as described above. Oral formulations (12 mg/kg, n=5
rats/group) of
cannabis extract in different carrier oils were administered via oral gavage,
and plasma
concentration of THC from 0 to 24 hours post-administration was measured as
described.
Values are expressed as mean SEM. The peak plasma concentration of THC
following
intravenous administration of cannabis extract in lipid-free solution was
measured as 864 194
ng/mL at 5 minutes (data not shown).
[000193] FIGS. 4A-4D summarize the pharmacokinetic parameters of THC
absorption. Oral
formulations (12 mg/kg, n=5 rats/group) of cannabis extract in different
carrier oils were
administered to rats as described above, and plasma concentration of THC from
1 to 8 hours
post-administration was measured as described. FIG. 4A shows the area under
the curve (AUC)
generated by plotting THC plasma concentration (in ng/mL) versus time in units
of hours (h).
FIG. 4B shows the maximum THC plasma concentration. FIG. 4C shows absolute THC
bioavailability; and FIG. 4D shows relative THC bioavailability. Values
expressed as mean
SEM. Statistical analysis was performed using unpaired two-tailed Student's t-
test and one-way
ANOVA, where appropriate.
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Example 2: Bioavailability of cannabinoids formulated with phospholipid-
containing extract
derived from krill
[000194] Cannabis extracts formulated in different lipid carriers and
phospholipid-containing
extracts derived from krill were administered to rats via oral gavage, and the
plasma
concentrations of exemplary cannabinoids THC and CBD were measured.
[000195] The study design and methods of analyses of the pre-clinical study
were the same as
disclosed in Example 1.
[000196] The dose groups were as follows:
= 1:1 THC:CBD IV in lipid-free solution (propyleneglycol-ethanol-sterile
water
(80:10:10 v/v/v); 4 mg/kg bw)
= 1:1 THC:CBD oral in MCT coconut oil (12 mg/kg bw)
= 1:1 THC:CBD oral in flax seed oil (TG-03) (12 mg/kg bw)
= 1:1 THC:CBD oral in krill oil (PL-03) (12 mg/kg bw)
TABLE 3: Pharmacokinetic parameters of CBD and THC after intravenous (IV)
bolus and
oral (gavage) administration of cannabis extract in various carrier oils
Formulation Clearance Vd AUCim Tmax Cmax fabs frel Flax
frel MCT
mL/h/lxg mL/kg 1)*(ng/mL) h ng/mL %
Intravenous administration
CBD IV 6991 26616 340 18 -
THC IV 10452 32764 231 18 -
Oral administration
CBD MCT 428 45 4.0 1.0 114 24 42 4 142 21
100
CBD TG-03 - 310 19$ 4.4 1.2 78 12 30 2$ 100
78 13
CBD PL-03 - 691 831 2.8 0.5 154 151 68 81 221
17* 178 42
THC MCT 1243 64 4.0 0.6 235 10 180 9 115
8 100
THC TG-03 - 1090 46 3.6 0.7 222 21 158 7 100
89 6
THC PL-03 - 1974 133* 3.6 0.7 381 51$ 285 19*
182 13* 162 18*
[000197] Table 3 shows the results from administering IV bolus of cannabinoids
in lipid-free
solution (4 mg/kg, n=4) and oral administration of cannabinoid formulations
(12 mg/kg,
n=5/group) via different carrier oils (krill oil, PL-03; flax seed oil, TG-03;
medium chain
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triglycerides from coconut oil, MCT) in rats. Data were taken from 0 to 8
hours post-dosage for
IV group and 1 to 8 hours for oral group. Values are expressed as mean SEM.
Statistical
analysis was performed using unpaired two-tailed Student's t-test and one-way
ANOVA, where
appropriate. * signifies p < 0.05 when compared to TG-03 (flaxseed) and MCT
oils; t signifies
p < 0.05 when compared to TG-03 (flaxseed) oil; $ signifies p < 0.05 when
compared to MCT
oil. Abbreviations: AUCrer, total area under the curve; CBD, cannabidiol;
Cmax, maximum
serum concentration; fabs, absolute bioavailability; frei, relative
bioavailability; THC,
tetrahydrocannabinol; T., time to reach maximum plasma concentration; Vd,
volume of
distribution.
[000198] FIG. 5 shows the plasma concentration-time profile of cannabidiol
(CBD)
administered to rats as described above. Oral formulations (12 mg/kg, n=5
rats/group) of
cannabis extract in different carrier oils were administered to rats, and the
plasma concentration
was measured from 0 to 24 hours post-administration. Values are expressed as
mean SEM.
The peak plasma concentration of CBD following intravenous administration of
cannabis
extract in lipid-free solution was measured as 1193 290 ng/mL at 5 minutes
(data not shown).
[000199] FIGS. 6A-6D summarize the pharmacokinetic parameters of CBD
absorption. Oral
formulations (12 mg/kg, n=5 rats/group) of cannabis extract in different
carrier oils were
administered to rats as described, and the plasma concentration of CBD from 1
to 8 hours post-
administration was measured. FIG. 6A shows the area under the curve (AUC)
generated by
plotting CBD plasma concentration (in ng/mL) versus time in units of hours
(h). FIG. 6B shows
the maximum CBD concentration. FIG. 6C shows absolute CBD bioavailability; and
FIG. 6D
shows relative CBD bioavailability. Values are expressed as mean SEM.
Statistical analysis
was performed using unpaired two-tailed Student's t-test and one-way ANOVA,
where
appropriate. * signifies p < 0.05 when compared to TG-03 (flax seed) and
medium chain
triglyceride (MCT) oils; 1- signifies p < 0.05 when compared to TG-03 (flax
seed) oil; $
signifies p < 0.05 when compared to MCT oil.
[000200] FIG. 7 shows the plasma concentration-time profile of
tetrahydrocannabinol (THC)
administered to rats as described above. Oral formulations (12 mg/kg, n=5
rats/group) of
cannabis extract in different carrier oils were administered to rats as
described and plasma
concentration of THC was measured from 0 to 24 hours post-administration.
Values are
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expressed as mean SEM. The peak plasma concentration of THC following
intravenous
administration of cannabis extract in lipid-free solution was measured as 864
194 ng/mL at 5
minutes (data not shown).
[000201] FIGS. 8A-8D summarize the pharmacokinetic parameters of
tetrahydrocannabinol
(THC) absorption. Oral formulations (12 mg/kg, n=5 rats/group) of cannabis
extract in different
carrier oils were administered to rats as described, and the plasma
concentration of THC was
measured from 1 to 8 hours post-administration. FIG. 8A shows the area under
the curve
(AUC) generated by plotting THC plasma concentration (in ng/mL) versus time in
units of
hours (h). FIG. 8B shows the maximum THC plasma concentration. FIG. 8C shows
absolute
THC bioavailability; and FIG. 8D shows relative THC bioavailability. Values
expressed as
mean SEM. Statistical analysis was performed using unpaired two-tailed
Student's t-test and
one-way ANOVA, where appropriate. * signifies p <0.05 when compared to TG-03
(flax
seed) and medium chain triglyceride (MCT) oils; $ signifies p < 0.05 when
compared to MCT
oil. In rats, bioavailability of cannabinoids was increased when emulsified in
carrier oils such
as krill and fish oil, as compared to MCT oil. This was observed as an
increased area under the
curve (AUC) and maximum concentration (Cmax).
Example 3: Bioavailability of Cannabinoids in Human Subjects
[000202] Cannabis extracts formulated in TG-03 (flax seed), MAG-03 (fish oil)
or PL-03
(krill oil), as well as medium chain triglyceride (MCT) as a control, are
administered to healthy
human volunteers. Subjects are dosed either once, or twice (an initial dose at
time = 0 hours,
and again at time = 12 hours) and the study is terminated at 24 hours.
Throughout the study,
blood is drawn from the subjects at regular intervals, and the pharmacokinetic
parameters of
cannabinoids are determined using standard methods (see Examples 1 and 2).
[000203] Cannabinoid formulations administered to subjects include
formulations with CBD
as the predominant cannabinoid, or formulations that include both CBD and THC.
[000204] Bioavailability is measured by standard techniques that assess plasma
concentration
over time to calculate the area under the curve (AUC), maximum concentration
(Cmax) and
time to maximum concentration (Tmax) following oral administration.
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[000205] In healthy volunteers, bioavailability of cannabinoids, variability
of bioavailability
between subjects, and variability caused by the effects of meals on
cannabinoid absorption are
improved when cannabinoids are formulated in flax seed, fish or krill oils,
when compared to
other carriers such as MCT oil. Improvements in bioavailability are observed
as increased
AUC, Cmax and Tmax. Improvement in variability and/or predictability of
cannabinoid
bioavailability is observed as reduced variance in AUC, Cmax and Tmax between
human
subjects, or by demonstrating that the pharmacokinetic parameters are
bioequivalent between
dosing in a fasted or fed state.
[000206] Bioavailability of cannabinoids administered orally to fasting
subjects is also
compared to bioavailability of cannabinoids administered orally to subjects
who have recently
consumed a high fat meal. When formulated in MCT oil, cannabinoids are more
rapidly
absorbed in fasting subjects compared to subjects who recently consumed a
fatty meal. This is
observed as a shorter Tmax in the fasting subject group. Overall
bioavailability is increased,
which is observed as a higher AUC and Cmax in the fatty meal group. The effect
of fasting
versus a high fat meal on bioavailability of cannabinoids formulated in TG-03,
MAG-03 or
PL-03 is assayed. Fasting subjects (for example, subjects who have fasted
overnight), and
subjects who have recently consumed a high fat meal (for example, in the 30
minutes prior to
administration of cannabinoid formulations), are administered CBD or CBD and
THC
formulated in TG-03, MAG-03 or PL-03, or MCT as a control.
[000207] Formulation of cannabinoids with TG-03, MAG-03 or PL-03 reduces
variation in
bioavailability with respect to a subject's recent eating history. Differences
between the Tmax,
AUC or Cmax in fasting individuals versus those that had consumed a high fat
meal are
reduced or absent, when comparing differences in the Tmax, AUC or Cmax of
subjects
administered cannabinoids formulated in MCT. Further, AUC, Cmax and Tmax of
subjects
administered cannabinoids formulated in TG-03, MAG-03 or PL-03 are improved
and less
variable compared to AUC, Cmax and Tmax of subjects administered cannabinoids
formulated
in MCT oil, in either the fasting or the high fat meal population. .
OTHER EMBODIMENTS
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[000208] While the invention has been described in conjunction with the
detailed description
thereof, the foregoing description is intended to illustrate and not limit the
scope of the
invention, which is defined by the scope of the appended claims. Other
aspects, advantages,
and modifications are within the scope of the following claims.
